A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif Tatyana I. Odintsova 1 , Alexander A. Vassilevski 2 , Anna A. Slavokhotova 1 , Alexander K. Musolyamov 2 , Ekaterina I. Finkina 2 , Natalia V. Khadeeva 1 , Eugene A. Rogozhin 2 , Tatyana V. Korostyleva 1 , Vitalii A. Pukhalsky 1 , Eugene V. Grishin 2 and Tsezi A. Egorov 2 1 Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia 2 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia Introduction To protect themselves against pathogens and pests, plants have developed a variety of mechanisms, includ- ing the creation of a physical barrier to limit pathogen spread and the production of antimicrobial compounds inhibiting pathogen growth and the colonization of plant tissues. Among the defensive compounds deployed by plants to combat infection are secondary metabolites, phytoalexins and phytoanticipins, reactive oxygen species and numerous proteins and peptides that exert inhibitory activity against invaders. Some antimicrobials are synthesized constitutively, whereas others are induced upon challenge with pathogenic microorganisms [1–5]. The first group contributes to preformed (basal) resistance, whereas the second group contributes to resistance activated in response to infec- tion or wounding by herbivores (induced resistance). Defense proteins produced by plants fall into two main categories according to their size: (a) proteins of > 100 amino acid residues and (b) smaller polypep- tides (< 100 amino acid residues), classified as pep- tides. Among the proteins implicated in plant defense, the so-called pathogenesis-related proteins play a key role [6]. They comprise a structurally and functionally heterogeneous groups of polypeptides, among them Keywords antifungal peptide; chitin-binding; cysteine motif; recombinant peptide; Triticum kiharae Correspondence T. Egorov, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16 ⁄ 10, 117997 Moscow, Russia Fax: +7 495 330 7301 Tel: +7 495 3364022 E-mail: ego@mx.ibch.ru Database The protein sequences reported in this paper have been submitted to the UniProtKB database under the accession number P85966 (Received 7 April 2009, revised 1 June 2009, accepted 5 June 2009) doi:10.1111/j.1742-4658.2009.07135.x Two forms of a novel antimicrobial peptide (AMP), named WAMP-1a and WAMP-1b, that differ by a single C-terminal amino acid residue and belong to a new structural type of plant AMP were purified from seeds of Triticum kiharae Dorof. et Migusch. Although WAMP-1a and WAMP-1b share similarity with hevein-type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described previously for plant AMPs, but is characteristic of the chitin-binding domains of cereal class I chitinases. An unusual substitution of a serine for a glycine residue in the chitin-binding domain was detected for the first time in hevein-like polypeptides. Recombinant WAMP-1a was successfully produced in Escherichia coli. This is the first case of high-yield production of a cysteine-rich plant AMP from a synthetic gene. Assays of recombinant WAMP-1a activity showed that the peptide possessed high broad-spectrum inhibitory activity against diverse chitin-containing and chitin-free patho- gens, with IC 50 values in the micromolar range. The discovery of a new type of AMP active against structurally dissimilar microorganisms implies divergent modes of action and discloses the complexity of plant–microbe interactions. Abbreviations AMP, antimicrobial peptide; CNBr, cyanogen bromide; Trx, thioredoxin. 4266 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS chitinases and 1,3-b-glucanases, proteinases and some other enzymes, proteinase and a-amylase inhibitors, thaumatin-like proteins and antimicrobial peptides (AMPs). AMPs form a highly evolved defense arsenal against pathogens that act in concert and form a ubiquitous tool of the plant innate immune system [5]. Most plant AMPs are cysteine-rich peptides con- taining an even number of cysteine residues, all of which are involved in the formation of intrachain disulfide bridges, providing their molecules with high structural stability [1,2,4]. Based on cysteine spacing motifs and 3D structures, several AMP families have been discriminated in plants, for example, thionins, defensins, hevein- and knottin-like peptides, and non- specific lipid-transfer proteins. Their mode of action includes disruption of pathogen membranes via spe- cific or nonspecific interactions with cell-surface groups [7,8]. Despite a conserved scaffold of mole- cules within the members of each family, there is considerable variation in amino acid sequences. There also exists considerable diversity in the processing and subcellular targeting of different AMP types [9]. New bioinformatics-based approaches have revealed an astonishing abundance of AMP-encoding genes in plant genomes, with hundreds of different genes being identified in the completely sequenced genomes of Arabidopsis and rice [10,11]. Such biodiversity ensures efficient defense against numerous invading and constantly evolving microorganisms. However, it remains unclear whether all genes identified in the genome are expressed and functional. In addition to being of fundamental significance in AMP research, they attract considerable attention as candidates for the genetic transformation of crops and as novel therapeutics. Hevein-type AMPs show structural similarity to he- vein, the 43-amino acid chitin-binding peptide isolated from the rubber tree Hevea brasiliensis [12], and com- prise the single hevein domain subfamily in a large group of chitin-binding proteins [13–16] that share a common property, the ability to bind chitin, a beta- 1,4-linked polymer of N-acetylglucosamine and related polysaccharides containing N-acetylglucosamine or N-acetylneuraminic acid. Because chitin does not occur in higher plants, but is a component of fungal cell walls and the exoskeleton of invertebrates, such as insects and nematodes, it has been hypothesized that chitin-binding proteins are involved in plant defense against microorganisms and pests. All known chitin- binding proteins contain a common structural motif of 30-45 amino acids with several cysteine and glycine residues at conserved positions named the chitin- binding domain, which is responsible for binding the carbohydrate. In addition to hevein, the members of this family are lectins, chitinases, some wound-induced proteins and a number of AMPs, later classified as he- vein-type. Although hevein-type AMPs share certain sequence homology, they differ in the number of disul- fide bonds. Most possess eight cysteine residues form- ing four disulfide bonds [13,14] and in this respect are close to the chitin-binding domains of class I ⁄ IV chitinases [15,16]. ‘Truncated’ variants with only six cysteine residues also occur [17–20]. Only a few ten- cysteine hevein-type AMPs have been described to date [21,22]. The functional significance of the additional cysteine residues of hevein-type AMPs remains to be elucidated. In a previous study, we used N-terminal sequences to identify several dozen novel AMPs from seeds of the wheat Triticum kiharae Dorof. et Migusch. [23]. In this study, we completely sequenced one of those novel peptides Tk-AMP-H1, in which the number of cysteine residues was corrected and which was renamed WAMP-1a. We show that it belongs to a new unique structural type of hevein-like AMP. We also report here high-yield heterologous production of WAMP-1a in Escherichia coli and assays of recombinant WAMP- 1a activity against diverse plant pathogens, such as chitin-containing and chitin-free fungi, and Gram- positive and Gram-negative bacteria. The amino acid sequence of a second peptide WAMP-1b, which differs from WAMP-1a by an additional C-terminal arginine residue, was also determined. Results Isolation and sequencing of WAMPs To purify WAMPs from T. kiharae seeds, the acidic extract of seeds was subjected to three-step chromato- graphic separation. Affinity chromatography on hepa- rin–Sepharose was the first step. The fraction eluted at 100 mm NaCl was further separated by size-exclusion chromatography and the peptide-containing fraction was then subjected to RP-HPLC (Fig. 1). The major peak during RP-HPLC (Fig. 1C) contained two pep- tides with measured monoisotopic molecular masses (by MALDI-TOF MS) of 4431.9 and 4588.1 Da. The peptides named WAMP-1a and WAMP-1b were puri- fied by rechromatography on the same column with a shallower acetonitrile gradient (10–40% B in 90 min, B: 80% acetonitrile containing 0.1% trifluoroacetic acid) (not shown). Both peptides were reduced and alkylated and their molecular masses became 5482.0 and 5638.5 Da for WAMP-1a and WAMP-1b, respec- tively, indicating the presence of 10 cysteine residues in T. I. Odintsova et al. Unique 10-cysteine antifungal peptide from wheat FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4267 both peptides. Alkylation of unreduced native WAMP peptides did not result in molecular mass changes, pointing to the involvement of all 10 thiol groups in the formation of five disulfide bridges. The complete amino acid sequences of reduced and alkylated peptides were determined by automated Edman degra- dation: WAMP-1a: AQRCGDQARGAKCPNCLCCG KYGFCGSGDAYCGAGSCQSQCRGC; WAMP-1b: AQRCGDQARGAKCPNCLCCGKYGFCGSGDAY CGAGSCQSQCRGCR. The peptides WAMP-1a and WAMP-1b consist of 44 and 45 residues, respectively, and are virtually iden- tical; WAMP-1b differs from WAMP-1a by an addi- tional C-terminal arginine residue. The measured molecular masses of the peptides are in good agree- ment with calculated values (4431.7 and 4587.8 Da for WAMP-1a and WAMP-1b, respectively), indicating the absence of modifications except disulfide bonds. By contrast, the other two known 10-Cys hevein-like pep- tides have a pyroglutamic acid at their N-termini [21,22]. WAMPs belong to a novel type of AMP. Despite sequence similarity with hevein and homologous pep- tides, they possess 10 cysteine residues and thus may be classified as 10-Cys hevein-like peptides with a unique previously unknown cysteine motif (Fig. 2). Only a few 10-Cys hevein-like peptides have been described to date, isolated from the bark of the trees Eucommia ulmoides Oliv. [21] and Euonymus ;europa- eus L. [22]. Amino acid sequence identity between WAMP-1a and Ee-CBP from E. europaeus, EAFP1 from Eu. ulmoides and hevein amounts to 50–56%. Although WAMPs, Ee-CBP and EAFP1 possess 10 cysteine residues, the cysteine motif in WAMPs differs remarkably from that of their 10-Cys homologues, indicating that WAMPs belong to a new structural type of hevein-like peptide (Fig. 2B,C). The uneven distribution of basic amino acids in WAMPs should be noted; except for a lysine residue at position 21, other basic residues are located in the N- and C-terminal regions of the molecules. Striking similarity with hevein-type domains of cereal class I chitinases, both in terms of amino acid sequences and cysteine patterns was noticed (Fig. 2A). High homology between a hevein-type AMP and the chitin-binding domain of the chitinase from the same source that act synergistically was also shown by Van den Bergh et al. [24]. The cysteine connectivities in WAMPs were deduced from sequence alignment with hevein. Of the 10 cyste- ines in WAMPs, eight (C1, C2, C4, C5, C6, C7, C8 and C9) are in an identical position in hevein, and consequently by homology form the same disulfide bridges (Fig 2B,C). Because WAMPs do not possess free thiol groups, two additional cysteines (C3 and C10) are concluded to be connected to each other. Consequently, it is reasonable to predict the arrange- ment of disulfide bonds in WAMPs as follows: C4–C19, C13–C25, C18–C32, C37–C41. An additional fifth disulfide bond is likely to be formed between C16 Fig. 1. Isolation of WAMP peptides. (A) Affinity chromatography of the acid-soluble extract from 5 g of T. kiharae seeds on a 5-mL HiTrap Heparin HP column. WAMP-containing fraction eluted at 100 m M NaCl is indicated by a gray box. (B) Size-exclusion chroma- tography on a Superdex Peptide HR 10 ⁄ 30 column of one half of the 100 m M NaCl fraction (see A). The WAMP-containing fraction is boxed. (C) RP-HPLC of the pooled WAMP-containing fraction (see B) on a Vydac C 18 column with a 60-min linear acetonitrile gradient (10–50% B, B: 80% acetonitrile in 0.1% trifluoroacetic acid). The fraction containing WAMPs is shown by an arrow. Unique 10-cysteine antifungal peptide from wheat T. I. Odintsova et al. 4268 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS and C44. This is supported by X-ray analysis of a class I chitinase from rice (Y. Kezuka, Y. Nishizawa, T. Watanabe & T. Nonaka, Nagaoka University of Technology, Japan; PDB accession number 2DKV). Binding of WAMPs to chitin The amino acids forming the chitin-binding site involved in carbohydrate recognition by hevein-type molecules deserve special attention (Fig. 2). In this site, several strictly conserved residues are present: serine 19, two glycines at positions 22 and 25 and three aro- matic amino acids at positions 21, 23 and 30, respec- tively (the numbering is according to hevein). In WAMPs from T. kiharae seeds, however, the con- served serine is replaced by a glycine. The chitin-bind- ing properties of both WAMP peptides were assayed in vitro. Purified peptides were applied to a chitin column and the bound fraction was eluted with 0.1% trifluoroacetic acid. RP-HPLC and mass measurements of unbound and bound fractions showed that both peptides eluted only in the bound fraction. Therefore, despite the serine to glycine substitution in the carbo- hydrate-binding site, WAMPs bind to chitin, although the affinity of this interaction and the precise impact of the Gly ⁄ Ser substitution are still to be investigated. Recombinant peptide production To provide sufficient material for functional investiga- tions, recombinant WAMP-1a was produced in a pro- karyotic expression system. Thioredoxin (Trx) was chosen as the fusion partner for expression, because it is known to ensure high yields of cystine-containing poly- peptides with native conformation and mask the unwanted toxic activity of the produced peptides [9,25]. A synthetic gene coding for WAMP-1a was prepared from oligonucleotides (Table 1) and cloned into pET-32b expression vector, and the resulting plasmid (pET-32b- M–WAMP-1a) was used to transform E. coli BL21 (DE3) cells. Trx–WAMP-1a fusion protein production and purification was followed by SDS ⁄ PAGE (Fig. 3). The chimeric protein was treated with cyanogen bromide (CNBr) and the recombinant WAMP-1a was purified by RP-HPLC (Fig. 3). The recombinant peptide had the same retention time and co-eluted with the native WAMP-1a when analyzed by analytical RP-HPLC, it also had the expected N-terminal amino acid sequence as determined by direct Edman sequencing. The molecular mass of the recombinant product obtained by MALDI MS was equal to the mass measured for the native WAMP-1a. The final yield of purified recombinant WAMP-1a was  8mgÆL )1 of bacterial culture. Antimicrobial activity of the recombinant WAMP-1a Testing of the biological activity of the recombinant peptide WAMP-1a against several fungi including deiteromycetes and ascomycetes showed marked inhibition of spore germination at micromolar concen- trations with IC 50 ranging from 5 to 30 lm depending on the fungus (Table 2). The highest inhibitory activity was achieved against Fusarium solani, Fusarium oxysporum and Bipolaris sorokiniana,IC 50 for these fungi was 5 lm, as low as reported for Ac-AMP2, one of the most potent antifungal peptides [17]. The Fig. 2. Sequence alignment of WAMP-1b with selected hevein- type antimicrobial peptides and chitin-binding domains of class I chitinases. Conserved cysteine residues are shaded in black. The conserved residues of the carbohydrate-binding site are marked with asterisks; the glycine residue in WAMP-1b that substitutes serine is boxed. (A) Manual alignment of WAMP-1b with chitin- binding domains of class I chitinases from cereals. GenBank acces- sion numbers are shown in parentheses for: wheat (Triticum aes- tivum, AAR11388), rye (Secale cereale, Q9FRV1), barley (Hordeum vulgare, P11955) and rice (Oryza sativa, 2DKV). Identical and similar residues are shaded in gray. (B) Hevein-type peptides: WAMP-1b from T. kiharae (P85966); Ee-CBP from the bark of E. europaeus (AAP35269); EAFP1 from the bark of Eu. ulmoides (P83596); hevein from H. brasiliensis (P02877); Ac-AMP2 from Amaranthus caudatus (P27275). (C) Disulfide bond arrangement in hevein-type AMPs. Cysteine residues found in all known hevein-like peptides are presented. Disulfide bonds in hevein are shown by solid line, an additional fifth disulfide in 10-Cys hevein-like peptides is shown by dashed line in WAMPs, short-dashed line in Ee-CBP and dashed-dotted line in EAFP1. T. I. Odintsova et al. Unique 10-cysteine antifungal peptide from wheat FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4269 morphological changes in two fungi, F. oxysporum and B. sorokiniana, in the presence of WAMP-1a were also examined. In F. oxysporum, inhibition of hyphal elon- gation and browning of hyphae were observed. In B. sorokiniana, the most pronounced changes occurred in spores; destruction and discoloration of spores were noted. The effect of the peptide on disease develop- ment caused by the oomycete Phytophthora infestans using potato tuber discs was also studied. Two differ- ent strains, the highly aggressive OSV 12 strain and the Pril 2 strain with low pathogenicity were tested. The peptide induced stable inhibition of disease devel- opment over 120 h of observation, followed by a slight decrease by 144 h in both instances. Complete inhibi- tion was not achieved at the concentrations tested. However, the degree of inhibition was higher with the less aggressive strain (Table 3). The peptide was also tested for inhibition of bacterial growth against both Gram-positive (Clavibacter michiganense ) and Gram-negative (Pseudomonas syringae and Erwinia carotovora) bacteria; the effect was most pronounced for the Gram-positive bacterium C. michiganense (Table 4). The antifungal activity of WAMP-1a is likely to be associated with its chitin-binding activity, whereas the inhibitory effect on the oomycete P. infe- stans and bacteria, which are devoid of chitin, implies the existence of some other mechanism. Discussion In this study, we purified and completely sequenced two novel highly homologous antimicrobial peptides, WAMP-1a and WAMP-1b, from T. kiharae seeds, which we previously discovered in this wheat species [23], and showed that they belong to a new structural type of plant AMP. Both peptides are almost identical, the WAMP-1b peptide possesses an additional arginine at the C-terminus. The WAMP peptides from T. kiha- rae seeds obviously belong to the hevein-type AMPs, as judged by sequence homology with hevein and homologous peptides and conserved location of eight cysteine residues and several other amino acids of the chitin-binding site (Fig. 2). The striking similarity between WAMPs and the chitin-binding domains of class I chitinases deserves special attention. Despite similarity with hevein and related proteins, WAMPs represent a new structural type of plant AMP with a specific 10-Cys motif. The characteristic feature of their molecular scaffold is the presence of an Fig. 3. Expression and purification of Trx–WAMP-1a fusion protein. RP-HPLC of the fusion protein Trx–WAMP-1a ( 0.5 mg) cleaved with CNBr on a Luna C 8 column. Fraction corresponding to WAMP- 1a is indicated with an arrow. The inset shows expression and puri- fication of Trx–WAMP-1a fusion protein as followed by SDS ⁄ PAGE (10%). Lane 1, molecular mass markers (LMW-SDS Marker Kit from GE Healthcare), the corresponding M r values are labeled in kDa; lane 2, whole-cell lysate of E. coli BL21(DE3) cells carrying the plasmid pET-32b-M–WAMP-1a before isopropyl b- D-thiogalactoside treatment; lane 3, induced with 0.2 m M isopropyl b-D-thiogalacto- side; lane 4, soluble protein fraction; lane 5, fusion protein purified by metal-affinity chromatography on TALON Superflow resin. Table 1. Synthetic oligonucleotides used for WAMP-1a gene con- struction. KpnI and BamHI restriction sites are underlined, the methionine codon is shown in bold, the stop codon is in bold and italics. Primer name Sequence Forward ACTG GGTACCATGGCTCAGCGTTGCGGTGAC 2f CAGGCTCGTGGTGCTAAATGCCCGAACTGCCTGTGCTGTG 3f GTAAGTACGGCTTCTGCGGTTCTGGTGACGCTTACTGTGG 4f CGCTGGTTCTTGCCAGTCTCAGTGCCGTGGTTGCTAG GGAT 1r TTTAGCACCACGAGCCTGGTCACCGCAACGCTGAGC 2r CGCAGAAGCCGTACTTACCACAGCACAGGCAGTTCG 3r GACTGGCAAGAACCAGCGCCACAGTAAGCGTCACCA Reverse GCTA GGATCCCTAGCAACCACGGCAC Table 2. Antifungal activity of WAMP-1a. IC 50 is the concentration necessary for 50% growth inhibition. Fungi IC 50 (lgÆmL )1 ) Bipolaris sorokiniana 5 Botrytis cinerea 20 Fusarium oxysporum 5 Fusarium solani 5 Fusarium verticillioides 30 Neurospora crassa 10 Unique 10-cysteine antifungal peptide from wheat T. I. Odintsova et al. 4270 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS additional fifth disulfide bond between C3 (Cys16) and C10 (Cys44), which is located differently from other known 10-Cys hevein-like peptides (Fig. 2B,C). This fifth disulfide bond brings together the N- and C-ter- minal regions of the polypeptide chain, enriched in basic amino acids (Arg3, Arg9, Lys12, Arg42 and Arg45 in WAMP-1b). We therefore suggest the exis- tence of a cluster of basic amino acid residues in WAMPs formed by the above-mentioned basic resi- dues of the N- and C-termini. However, elucidation of the 3D structure of WAMPs is necessary to confirm this hypothesis. The second structural peculiarity of WAMPs that discriminates these peptides from all known chitin-binding polypeptides is the unique struc- ture of the chitin-binding site, in which a conserved serine residue at position 20 is substituted for glycine, although three aromatic residues (Tyr22, Phe24 and Tyr31) are well conserved. To the best of our knowledge, this is the first communication on such a replacement in the chitin-binding site. Analysis of chi- tin-binding properties of WAMPs by in vitro assays showed that both peptides bind chitin, demonstrating that the serine ⁄ glycine substitution is not crucial for binding, although its precise role in the efficiency of binding remains to be explored. Unusual structural characteristics of WAMPs sug- gested unique biological properties, however, limited amounts of the peptides recovered from T. kiharae seeds were insufficient for large-scale assays of their biological activity. To produce the target peptide in a correctly folded soluble form and eliminate possible toxic effects on the host cells, thioredoxin, a natural soluble component of E. coli cells, was chosen as a fusion partner. Preliminarily, the synthetic gene encoding WAMP-1a was constructed. As a result, the biologically active 10-Cys recombinant peptide WAMP-1a was successfully produced in E. coli with high yields (8 mgÆL )1 of culture). To the best of our knowledge, for the first time, a synthetic gene con- struction was used to generate a 10-Cys AMP in E. coli. This approach is indispensable for the rapid production of AMPs with unknown functions avoid- ing time-consuming gene cloning. Moreover, it effec- tively allows introducing codons optimized for E. coli. The biological activity of the recombinant WAMP-1a was assayed against fungi, oomycetes and bacteria. The results showed that the peptide has broad inhibitory activity both against chitin-contain- ing and chitin-free pathogens. The peptide not only inhibited spore germination of the deiteromycetes and ascomycetes tested, but also caused morphological changes in the fungi. The activity of the peptide both against chitin-containing and chitin-free pathogens may result from the unique structural features of WAMPs detailed above. We speculate that the inhibi- tion of morphogenesis in chitin-containing fungi is associated with its chitin-binding activity, whereas the effect on bacteria may result from interactions between the cluster of basic amino acid residues in WAMP molecules and negatively charged phospholip- ids of the bacterial membranes, as postulated for membrane-active amphiphilic AMPs. The positive charge ensures accumulation at polyanionic microbial cell surfaces that contain acidic polymers, such as lipopolysaccharide and cell-wall-associated teichoic acids in Gram-negative and Gram-positive bacteria, respectively. By insertion into the membrane, amphi- philic AMPs disrupt the integrity of the bilayer through membrane thinning, transient poration and ⁄ or disruption of the barrier function, or translo- cate across the membrane and act on internal targets [7,26]. In summary, two novel hevein-type chitin-binding AMPs, WAMP-1a and WAMP-1b, were purified from T. kiharae seeds and sequenced. The peptides consist of 44 and 45 amino acids, respectively, and differ by a single amino acid residue at the C-terminus. The WAMP peptides belong to a new structural type of hevein-like AMP with sequence similarity to chitin- binding domains of cereal class I chitinases. Testing of the biological activities of the recombinant peptide Table 4. Antibacterial activity of WAMP-1a. Peptide concentration (lgÆ50 lL )1 ) a Inhibition zone in cm including the size of the peptide application zone b Clavibacter michiganense Erwinia carotovora Pseudomonas syringae 10 1.7 (3.6) 1.5 (3.4) 1.3 (1.4) 5 1.5 (3.2) 1.3 (2.7) 1.2 (1.2) 2.5 1.3 (3.0) 1.1 (1.1) 0.9 (1.0) a Sample volume 50 lL. b Size of the peptide application zone 0.5 cm. The size of the inhibition zone caused by claforan is shown in parentheses. Table 3. The effect of WAMP-1a on Phytophthora infestans dis- ease development. Peptide concentrations in micromoles are shown in brackets. Strain 96 h 120 h 144 h PRIL 2 +++ (5.0) +++ (5.0) ++ (5.0) OSV 12 ++ (5.0) + (5.0) no effect OSV 12 ++ (10) ++ (10.0) + (10.0) T. I. Odintsova et al. Unique 10-cysteine antifungal peptide from wheat FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4271 WAMP-1a expressed from a synthetic gene in E. coli showed potent antifungal and antibacterial effects at micromolar concentrations. The discovery of WAMPs expands our knowledge on the molecular diversity of AMPs produced by plants to combat pathogenic microorganisms. Experimental procedures Biological material and chemicals Seeds of T. kiharae Dorof. et Migush. (Poaceae, Magno- liophyta) were obtained from the collection of the Institute of General Genetics of the Russian Academy of Sciences (Moscow, Russia). Fungi and bacteria, F. solani VKM F- 142, F. verticillioides VKM F-670, F. oxysporum TSA-4, B. cinerea VKM F-85, Neurospora crassa VKM F-184, Ps. syringae VKM B-1546, C. michiganense subsp. michigan- ense VKM Ac-1144 and Er. carotovora subsp. carotovora VKM B-1247 were obtained from the All-Russian Collec- tion of Microorganisms (Pushchino, Russia). The fungus B. sorokiniana, strain 6 ⁄ 10 was from the Timiryazev Agricul- tural Academy (Moscow, Russia). The oomycete P. infestans strains Pril 2 and OSV 12 were from the Institute of Plant Protection (Priluki, Minsk District, Bellorussia). E. coli BL21(DE3) strain and the expression vector pET-32b (Nov- agen, Madison, WI, USA) were purchased from Rusbiolink (Moscow, Russia). Restriction enzymes, T4 DNA ligase, PCR reagents, PCR clean-up system and DNA purification system were from Promega (Madison, WI, USA). TALON Superflow Metal Affinity Resin (Clontech, Mountain View, CA, USA) was used for affinity chromatography. Bacterial cultures were grown using the safety recommendations from All-Russian Collection of Microorganisms. Chemicals were from Sigma-Aldrich (St. Louis, MO, USA), Merck (Darm- stadt, Germany) and UV-grade acetonitrile was from Cryochrom (St Petersburg, Russia). 4-Vinylpyridine (Sigma- Aldrich) was vacuum distilled under argon. Isolation of WAMPs Isolation of WAMPs from T. kiharae seeds mainly fol- lowed the earlier developed procedure [23]. Briefly, flour was extracted with 5 volumes of mixture of 1% trifluoro- acetic acid, 1 m HCl, 5% HCOOH and 1% NaCl in the presence of the proteinase inhibitor cocktail for plant cell extracts (Sigma-Aldrich). Extracted proteins and peptides were precipitated with acetone. The pellet was dissolved in 0.1% trifluoroacetic acid, desalted by RP-HPLC and dried on a SpeedVac concentrator, whereupon it was subjected consecutively to three types of HPLC: affinity, size-exclu- sion and reversed-phase. First, the desalted fraction was solubilized in 10 mm Tris ⁄ HCl, pH 7.2 (buffer A) and subjected to affinity chromatography on a 5-mL HiTrap Heparin HP column (GE Healthcare, Little Chalfont, UK) equilibrated with buffer A. After elution of the unad- sorbed fraction, proteins and peptides were eluted with a step-wise NaCl gradient in buffer C: 50, 100 and 500 mm NaCl at a flow rate of 1 mLÆmin )1 . Proteins and peptides were detected at 280 nm. The obtained fractions were desalted and dried as described above. The fraction eluted at 100 mm NaCl during affinity chromatography was sep- arated by size-exclusion chromatography on a Superdex Peptide HR 10 ⁄ 30 column (GE Healthcare). Proteins and peptides were eluted with 5% CH 3 CH in 0.05% trifluoro- acetic acid at a flow rate of 15 mLÆh )1 and detected at 214 nm. The peptide fraction was further separated by RP-HPLC on a Vydac 218TP54 C 18 column (4.6 · 250 mm; Separations Group, Hesperia, CA, USA) with a 60-min linear acetonitrile gradient (10–50% B, B: 80% acetonitrile in 0.1% trifluoroacetic acid) at a flow rate of 1mLÆmin )1 and detection at 214 nm. Reduction and alkylation of peptides Reduction with dithiothreitol and alkylation with 4-vinyl- pyridine were accomplished essentially as earlier described [23]. Shortly, the protein was dissolved in 20 lL of 0.5 m Tris ⁄ HCl buffer, pH 7.6, containing 6 m guanidine hydro- chloride and 2 mm EDTA (disodium salt). One microliter of freshly prepared 1.4 lm aqueous dithiothreitol solution was added to the mixture. The reaction was allowed to proceed under nitrogen for 4 h at 40 °C. After reduction, 2 lL of 50% 4-vinylpyridine in 2-propanol were 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% trifluoroacetic acid and separated by RP-HPLC on a Vydac column as described above. The number of cysteine residues was estimated from the mass difference between the reduced and alkylated and nonalkylated polypeptide. Analytical methods Peptides were analyzed by MALDI-TOF MS. Mass spec- tra were acquired on a model Ultraflex MALDI-TOF- TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Calibration was performed using a Proteo- Mass peptide and protein MALDI-MS calibration kit (mass range 700–66 000 Da; Sigma-Aldrich). Molecular masses were determined in linear or reflector positive-ion mode using samples prepared using the dried-droplet method with a-cyano-4-hydroxycinnamic acid (10 mgÆmL )1 in 50% acetonitrile with 0.1% trifluoroacetic acid) matrix (Sigma-Aldrich). Amino acid sequences were determined by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems, Foster City, CA, USA). Unique 10-cysteine antifungal peptide from wheat T. I. Odintsova et al. 4272 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS Absorption spectra were recorded on a Hitachi U-3210 spectrophotometer (Hitachi, Tokyo, Japan). Polypeptide concentrations were determined using molar extinction coefficients at 280 nm (e 280 ) calculated using the gpmaw program (Lighthouse Data, Odense, Denmark; http://www. gpmaw.com/): 3160 m )1 Æcm )1 for WAMP-1a, 17 220 m )1 Æcm )1 for the fusion protein Trx–WAMP-1a. Chitin-binding assay The peptide (2 nmol) was dissolved in 50 mm NH 4 HCO 3 , pH 7.8, and loaded onto a chitin column (0.5 mL) equili- brated with the same buffer. Elution of the unadsorbed fraction was performed until the absorbance of the eluate at 280 nm reached the value of 0.01, the bound fraction was eluted with 0.1% trifluoroacetic acid. Both fractions were analyzed by MS and RP-HPLC. Expression vector construction Antimicrobial peptide expression was achieved essentially by following a previously elaborated procedure [25]. The DNA sequence encoding WAMP-1a was constructed from a number of synthetic oligonucleotides (Table 1) using the PCR technique. The target PCR fragment was amplified using a forward primer containing a KpnI restriction site and a Met codon for CNBr cleavage, and a reverse pri- mer containing a BamHI restriction site and a stop codon. The PCR fragment was gel purified, digested by suitable restriction enzymes and cloned into the expression vector pET-32b (Novagen) to produce pET-32b- M–WAMP-1a. The resulting construct was checked by sequencing. Fusion protein production and purification E. coli BL21(DE3) cells transformed with the expression vector pET-32b-M–WAMP-1a were cultured at 37 °Cin Luria–Bertani medium containing 100 lgÆmL )1 ampicillin to a culture density of D 600 = 0.4–0.8. Expression was induced by adding isopropyl b-d-thiogalactoside to a con- centration of 0.2 mm. Cells were cultured at room tempera- ture (24 °C) overnight (16 h) and harvested (centrifuged for 20 min at 5000 g). The cell pellet was resuspended in the start buffer for affinity chromatography (1 g of wet weight cells in 10 mL of 300 mm NaCl, 20 mm Tris ⁄ HCl buffer, pH 7.5) and ultrasonicated. Lysed cells were centrifuged for 15 min at 20 000 g to remove all insoluble particles. The supernatant was applied to a preliminarily equilibrated TALON Superflow resin (volume of 3 mL; Clontech) and the fusion protein (Trx–WAMP-1a) was purified according to the protocol supplied by the manufacturer (washed with the buffer containing 5 mm imidazole, 500 mm NaCl, 5% glycerol, 0.1% Triton X-100, 20 mm Tris ⁄ HCl, pH 7.5, and eluted with the final elution buffer containing 150 mm imid- azole, 300 mm NaCl, 20 mm Tris ⁄ HCl, pH 7.5). Fusion protein cleavage and target peptide isolation The hybrid protein was quickly desalted on a Jupiter C 5 column (4.6 · 150 mm; Phenomenex, Torrance, CA, USA), using a step of acetonitrile concentration (0–70%) in 0.1% trifluoroacetic acid. The collected fusion protein was dried on a vacuum concentrator at room temperature and dissolved in 0.1 m HCl. Protein cleavage with CNBr was performed overnight (16 h) at room temperature (24 °C) in the dark, with a protein-to-CNBr molar ratio of 1 : 1000. The solvent and excess CNBr were removed on a SpeedVac concentrator. Recombinant WAMP-1a was purified by RP-HPLC on a Luna C 8 column (4.6 · 150 mm; Phenomenex) in a linear gradient of acetonitrile (5– 25% in 30 min, 25–60% in 10 min) in 0.1% trifluoroacetic acid; detection was performed by measuring effluent absor- bance at 280 nm. The purity of the target peptide was checked by MS, as well as by N-terminal sequencing, and the concentration was measured by optical absorption at 280 nm. Chromatographic retention times of recombinant and natural WAMP-1a were compared by co-injecting samples onto a Vydac C 18 column and running a shallow acetonitrile gradient. Antifungal assays The antifungal activity of the peptides was tested against several fungi using microtiter-plate assays essentially as described previously [27]. Wells were filled with 10 lLof twofold serial dilutions of the peptide and mixed with 90 lL half-strength potato–glucose broth containing  10 4 sporesÆmL )1 . The inhibition of spore germination was evaluated by measuring the absorbance at 620 nm. Morphological changes were recorded using a light micro- scope. The biological activity of peptides was also assayed by estimating the degree of inhibition of the oomycete P. infe- stans development on potato tuber discs. Two potato tuber discs of similar size were placed in each Petri dish. Peptide samples were mixed with 50 lL of zoosporangium suspen- sion in distilled water (2 · 10 4 zoosporangiaÆmL )1 ) to a final peptide concentration of 1.25–20 lm and incubated at 20 °C for 2 h. The peptide sample was applied to the center of each potato tuber disc. Potato discs infected with zoospo- rangium suspension without peptide served as controls. Petri dishes with infected potato tuber discs were incubated at 20 °C for 120 h. The severity of the disease was assayed 96, 120 and 144 h after inoculation by measuring the infected area of each disc and scored from 0 to ++++, with 0 denoting the absence of inhibition compared with controls, T. I. Odintsova et al. Unique 10-cysteine antifungal peptide from wheat FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4273 + denoting low inhibitory activity (disease development 20– 40%), ++ designating moderate inhibitory activity (disease development 10–20%,), +++ denoting strong inhibition (disease development below 10%), and ++++ represent- ing complete inhibition (no disease symptoms are observed). Ten discs were analyzed in each of three independent experi- ments. The morphological changes in the fungi were also recorded using a light microscope. Antibacterial assays The antibacterial activity of peptides was assayed against several Gram-positive and Gram-negative bacteria using radial diffusion assay. Petri dishes with Luria–Bertani agar were seeded with test bacteria. The peptide solutions (50 lL) were applied to the wells (5 mm in diameter) punched into the agar, and the Petri dishes were incubated at room temperature for 24–48 h. The antibacterial activity was evaluated by the size of the inhibition zone formed around the wells with the peptide solution. The antibiotic claforan and sterile water were used as controls. Acknowledgements This work was supported in part by the Biodiversity Program of the Russian Academy of Sciences and grants from the Russian Foundation for Basic Research (no. 08-04-00783 and no. 09-04-00250). We also thank Bert Billen (KU Leuven, Belgium) for help with manuscript preparation. References 1 Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, De Samblanx GW & Osborn RW (1997) Antimicro- bial peptides from plants. Crit Rev Plant Sci 16, 297– 323. 2 Garcia-Olmedo F, Molina A, Alamillo JM & Rodri- guez-Palenzuela P (1998) Plant defense peptides. Bio- polymers 47, 479–491. 3 Selitrennikoff CP (2001) Antifungal proteins. Appl Envi- ron Microbiol 67, 2883–2894. 4 Garcia-Olmedo F, Rodriguez-Palenzuela P, Molina A, Alamillo JM, Lopez-Solanilla E, Berrocal-Lobo M & Poza-Carrion C (2001) Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence. FEBS Lett 498, 219–222. 5 Manners JM (2007) Hidden weapons of microbial destruction in plant genomes. Genome Biol 8, 225, doi:10.1186/gb-2007-8-9-225. 6 Sels J, Mathys J, De Coninck BM, Cammue BP & De Bolle MF (2008) Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiol Biochem 46, 941–950. 7 Shai Y (2002) Mode of action of membrane active anti- microbial peptides. Biopolymers 66, 236–248. 8 Thevissen K, Ferket KK, Francois IE & Cammue BP (2003) Interactions of antifungal plant defensins with fungal membrane components. Peptides 24, 1705–1712. 9 Vassilevski AA, Kozlov SA & Grishin EV (2008) Anti- microbial peptide precursor structures suggest effective production strategies. Recent Pat Inflamm Allergy Drug Discov 2, 58–63. 10 Silverstein KA, Graham MA, Paape TD & VandenBosch KA (2005) Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol 138, 600–610. 11 Silverstein KA, Moskal WA Jr, Wu HC, Underwood BA, Graham MA, Town CD & VandenBosch KA (2007) Small cysteine-rich peptides resembling antimi- crobial peptides have been under-predicted in plants. Plant J 51, 262–280. 12 Van Parijs J, Broekaert WF, Goldstein IJ & Peumans WJ (1991) Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex. Planta 183, 258–264. 13 Koo JC, Lee SY, Chun HJ, Cheong YH, Choi JS, Kawabata S, Miyagi M, Tsunasawa S, Ha KS, Bae DW et al. (1998) Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity. Biochim Biophys Acta 1382, 80–90. 14 Li SS & Claeson P (2003) Cys ⁄ Gly-rich proteins with a putative single chitin-binding domain from oat (Avena sativa) seeds. Phytochemistry 63, 249–255. 15 Raikhel NV, Lee HI & Broekaert WF (1993) Structure and function of chitin-binding proteins. Annu Rev Plant Physiol Plant Mol Biol 44, 591–615. 16 Beintema JJ (1994) Structural features of plant chitinas- es and chitin-binding proteins. FEBS Lett 350, 159–163. 17 Broekaert WF, Marien W, Terras FR, De Bolle MF, Proost P, Van Damme J, Dillen L, Claeys M, Rees SB, Vanderleyden J et al. (1992) Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine ⁄ glycine-rich domain of chitin-binding proteins. Biochemistry 31, 4308–4314. 18 Huang X, Xie W & Gong Z (2000) Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba. FEBS Lett 478, 123–126. 19 Nielsen KK, Nielsen JE, Madrid SM & Mikkelsen JD (1997) Characterization of a new antifungal chitin-bind- ing peptide from sugar beet leaves. Plant Physiol 113, 83–91. 20 Lipkin A, Anisimova V, Nikonorova A, Babakov A, Krause E, Bienert M, Grishin E & Egorov T (2005) An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds. Phytochemistry 66, 2426–2431. Unique 10-cysteine antifungal peptide from wheat T. I. Odintsova et al. 4274 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 21 Huang RH, Xiang Y, Liu XZ, Zhang Y, Hu Z & Wang DC (2002) Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv. FEBS Lett 521, 87–90. 22 Van den Bergh KP, Proost P, Van Damme J, Coosemans J, Van Damme EJ & Peumans WJ (2002) Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.). FEBS Lett 530, 181–185. 23 Egorov TA, Odintsova TI, Pukhalsky VA & Grishin EV (2005) Diversity of wheat anti-microbial peptides. Peptides 26, 2064–2073. 24 Van den Bergh KP, Rouge P, Proost P, Coosemans J, Krouglova T, Engelborghs Y, Peumans WJ & Van Damme EJ (2004) Synergistic antifungal activity of two chitin-binding proteins from spindle tree (Euonymus europaeus L.). Planta 219, 221–232. 25 Shlyapnikov YM, Andreev YA, Kozlov SA, Vassilevski AA & Grishin EV (2008) Bacterial pro- duction of latarcin 2a, a potent antimicrobial peptide from spider venom. Protein Expr Purif 60, 89–95. 26 Hancock RE & Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24, 1551–1557. 27 Broekaert WF, Terras FRG, Cammue BPA & Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69, 55–59. T. I. Odintsova et al. Unique 10-cysteine antifungal peptide from wheat FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4275 . CGCTGGTTCTTGCCAGTCTCAGTGCCGTGGTTGCTAG GGAT 1r TTTAGCACCACGAGCCTGGTCACCGCAACGCTGAGC 2r CGCAGAAGCCGTACTTACCACAGCACAGGCAGTTCG 3r GACTGGCAAGAACCAGCGCCACAGTAAGCGTCACCA Reverse GCTA GGATCCCTAGCAACCACGGCAC Table. A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif Tatyana I. Odintsova 1 , Alexander A. Vassilevski 2 ,