The γ-Core Motif Peptides of AMPs from Grasses Display Inhibitory Activity against Human and Plant Pathogens

Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.


Introduction
Pathogenic microorganisms cause diseases in plants and animals. About 80,000 plant diseases have been reported in the world [1]. The overwhelming number of plant pathogens (over 80%) belong to phytopathogenic fungi [2]. Virtually all plants are hosts to particular fungal pathogens. The disease symptoms caused by fungi vary and include spots, wilts, rots, blight, and cankers. The ubiquitous Fusarium genus comprises approximately 300 species/species complexes that cause diseases of almost all economically important plants [3]. The most common diseases induced by these pathogens are vascular wilts and root rots. In addition to reducing crop yields, the Fusarium fungi produce mycotoxins, toxic secondary metabolites, which contaminate food and thus are harmful to human health. Some of the Fusarium species belong to opportunistic human pathogens affecting individuals with impaired immunity [4].
Pathogenic bacteria pose another threat to plants' health. Of the more than 7000 bacterial species, about 150 species cause diseases in plants [5]. The disease symptoms are diverse: galls, leaf spots, wilts, blights, soft rots, scabs, and cankers. The bacterial species that are pathogenic to plants belong to a number of genera, including Pectobacterium, Pseudomonas, and Clavibacter [6]. 2

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The economic consequences of plant diseases for agriculture are yield losses amounting to 50% in epiphytotic years and a reduction in the quality of agricultural production due to its contamination with pathogen-derived toxic secondary metabolites [7]. The use of fungicides reduces yield failure; however, this has a negative impact on the environment and stimulates the emergence of resistant strains of pathogens. With the world's constantly growing population, the need for new alternative crop protection products is steadily increasing.
About 200 fungal species cause diseases (mycoses) in humans. The genus Candida consists of approximately 200 yeast species. Of them, about a dozen are associated with the vast majority of human infections caused by fungal pathogens [8]. In the last two decades, the incidence of human diseases caused by Candida species has been steadily increasing [9]. Cryptococcus neoformans is another important opportunistic human pathogen [10]. In severe cases, it causes cryptococcal meningitis, an extremely serious infection with high mortality rates. With considerable progress in cancer treatment and organ transplantation, the number of patients with profound immunosuppression, which are at risk of acquiring lifethreatening fungal infections, is constantly increasing. The therapeutic efficacy and clinical applicability of the available antifungal agents is limited by their toxicity, narrow activity spectrum, inability to fully eliminate infection, and resistance development. The search for novel alternative antifungals with enhanced potency, minimal toxicity, broad-spectrum activity and effective tissue penetration has become a priority.
Antimicrobial peptides (AMPs) are ubiquitous proteinaceous defense compounds, which comprise an essential part of the immune system of plants, animals, and microorganisms [11][12][13]. The array of AMPs that are present permanently in each plant species and those synthesized in response to pathogen invasion form the basis for the so-called innate (or non-specific) immunity. Plant AMPs are mainly cysteine-rich molecules, whose structure is stabilized by three to five disulfide bonds [14][15][16]. They differ significantly in their amino acid sequences and 3D structure resulting in a multifaceted mode of action and diverse host range. Disturbance of the membranes of pathogenic microorganisms is believed to be the predominant mechanism of microbe killing. As mentioned above, the development of resistance to conventional antibiotics in pathogens and the negative environmental impact of chemicals used for plant protection require the search for new alternative means of fighting human, animal, and plant diseases. Plant AMPs, which are natural antibiotics acting rapidly on a wide range of pathogens and showing a low incidence of resistance development, seem excellent candidates for creating new drugs and plant disease control agents. However, the size of some plant AMPs and their disulfidelinked structures, resulting in high cost of production, are a barrier to their widespread use. To overcome these limitations, short peptide fragments corresponding to the sequences in the intact AMP molecules that are responsible for the antimicrobial activity can be regarded as cost-effective templates for creating novel antimicrobial agents with a view to their use in agriculture and medicine.
Studies on the structure-function relationship in plant AMPs have shown that antimicrobial activity is associated with a specific part of the cysteine-rich peptide (CRP) molecule, the so-called γ-core. γ-Core was defined as a region with a GXCX 3-9 C signature adopting a β-hairpin conformation in the AMP's 3D structure [17]. In our previous work, we explored the antimicrobial activity of the γ-core motif peptides of a number of Solanum lycopersicum L. CRPs involved in response to infection with the fungus Fusarium oxysporum and the biogenic resistance inducers [18]. We showed that the γ-cores of several tomato CRPs possess potent antimicrobial activity against plant and human pathogens. In this work, we continue our research on antimicrobial properties of the γ-core motif peptides from plant AMPs. This time, we focused on the AMPs of the wheat species Triticum kiharae Dorof. et Migush., which is highly resistant to plant pathogens and is, therefore, potentially a rich source of novel antimicrobials. In this study, we have identified the γ-core motifs in AMPs predicted in the transcriptomes of T. kiharae infected with F. oxysporum and treated with F. sambucinum elicitors, produced them by solid-phase synthesis and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. As a result, we discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides derived from the 4-Cys-containing defensin-like (DEFL) peptides, whose role in plant immunity has remained unknown. In addition to identification of the determinants of the antimicrobial properties of plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to be used in control of plant pathogens.

Design of γ-Core Peptides
Seventeen AMPs belonging to the families of DEFLs, snakins, non-specific lipidtransfer proteins (nsLTPs), thionin-and knottin-like peptides, which were identified in wheat by transcriptome or amino acid sequencing [19][20][21][22], and peptide HvDEFL4-1 of barley discovered by BLAST search (GenBank: BAJ88142.1) were selected for the synthesis of the γ-core motif peptides and their truncated variants ( Figure 1). The γ-core peptides and their derivatives were produced by chemical synthesis and purified by high-performance liquid chromatography. therefore, potentially a rich source of novel antimicrobials. In this study, we have identified the γ-core motifs in AMPs predicted in the transcriptomes of T. kiharae infected with F. oxysporum and treated with F. sambucinum elicitors, produced them by solid-phase synthesis and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. As a result, we discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides derived from the 4-Cys-containing defensin-like (DEFL) peptides, whose role in plant immunity has remained unknown. In addition to identification of the determinants of the antimicrobial properties of plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to be used in control of plant pathogens.

Design of γ-Core Peptides
Seventeen AMPs belonging to the families of DEFLs, snakins, non-specific lipid-transfer proteins (nsLTPs), thionin-and knottin-like peptides, which were identified in wheat by transcriptome or amino acid sequencing [19][20][21][22], and peptide HvDEFL4-1 of barley discovered by BLAST search (GenBank: BAJ88142.1) were selected for the synthesis of the γ-core motif peptides and their truncated variants ( Figure 1). The γ-core peptides and their derivatives were produced by chemical synthesis and purified by high-performance liquid chromatography.

4-Cys DEFLs
Four synthetic peptides were designed on the basis of the sequences of 4-Cys-containing DEFLs, which have a cysteine signature as follows: C-X{3,5}-C-X{8,17}-C-X{4,6}-C ( Figure 1). When compared to the classical defensins, the biological functions of 4-Cys-containing DEFLs in wheat physiology remain obscure [19], therefore, testing the antimicrobial properties of these CRPs seemed a reasonable way to elucidate their role in defense. The selected DEFLs included DEFL4-4, DEFL4-8, DEFL4-20 and DEFL4-37. All but one 4-Cys DEFL showed sequence similarity to uncharacterized proteins of the diploid relatives of hexaploid wheat-Aegilops tauschii Coss. and Triticum urartu Thumanjan ex Gandilyan [19]. DEFL4-4 displayed no sequence similarity to known proteins. All selected 4-Cys DEFLs except DEFL4-37 possessed a γ-core motif and were predicted to be AMPs. The expression profiling showed that the DEFLs 4-4, 4-8 and 4-20 were up-regulated by the elicitors and/or fungal infection suggesting their involvement in the activation of the immune response by the resistance inducers and/or pathogen [19]. Conversely, DEFL4-37 was down-regulated, pointing to its role as a negative regulator of the immune response in wheat. In addition to the full-length DEFL4 γ-core peptides, their shortened versions were also synthesized ( Figure 1). Truncated variants of the γ-core peptides with high antimicrobial activity are preferable for practical applications due to a low cost of production. These truncated versions of DEFL4 γ-core peptides represented the loops between the second and the third cysteine residues. The avoidance of highly reactive cysteine residues in the peptide sequence is another beneficial characteristic for the production of chemically stable antimicrobials. The γ-core peptide DEFL4-20 86-110 was the longest of all synthesized peptides; its sequence spanned the 4-Cys motif of DEFL4-20 including the γ-core. Its truncated variant DEFL4-20 92-102 contained only the loop between the second and the third cysteines. The peptide fragment DEFL4-4 46-57 also represented the loop between the second and the third cysteine in DEFL4-4 ( Figure 1). The fragment pairs DEFL4-8 82-94 /DEFL4-8 83-93 and DEFL4-37 90-102 /DEFL4-37 91-101 corresponded to the above-mentioned loops in DEFL4-8 and DEFL4-37 with or without the adjacent cysteines, respectively. For comparison, a peptide fragment HvDEFL4-1 67-77 of Hordeum vulgare L. HvDEFL4-1 with the same cysteine signature as the wheat 4-Cys DEFLs was also synthesized. Its sequence corresponded to the loop between the second and the third cysteines in HvDEFL4-1 ( Figure 1).
In total, 20 γ-core peptides from 17 wheat AMP-like peptides and one peptide from barley were synthesized for antimicrobial activity assays.

Physicochemical Properties of Synthetic Peptides
The physicochemical properties of wheat and barley synthetic peptides are summarized in Table 1. The peptides contain from 11 to 25 amino acid residues. All of them are cationic. The pI (isoelectric point) values vary from 7.93 in TkThi1 96-109 to 11.71 in DEFL4-37 91-101 . The net charge is in the range from +1 to +8. It has been generally acknowledged that the positive charge is essential for the initial electrostatic interaction of the peptide with the anionic cell surfaces of pathogenic microorganisms [24]. The aliphatic index, which reflects protein thermostability, varies from 0 in DEFL1-16 65-82 and DEFL1-32 55-68 , since these peptides have no Ala, Val, Leu and Ile residues, to 53.08 in TkLTP2.25 50-62 (Table 1). Thus, the structure of TkLTP2.25 50-62 is predicted to be the most stable under a wide range of temperature regimes. The ratio of hydrophobic amino acid residues ranges from 11 in DEFL1-23 65-82 to 55 in DEFL4-8 83-93 , DEFL4-20 92-102 and DEFL4-37 91-101. The average ratio of hydrophobic residues in AMPs is about 41.5%. Higher hydrophobicity facilitates the insertion of the peptide into the lipids of the microbial membranes. However, too high hydrophobicity makes the peptide insoluble in aqueous environments. The GRAVY (Grand Average of Hydropathy) index is positive for TkThi1 96-109 and Tk-AMP-K2 10-23 and negative for all other peptides (Table 1). Negative values mean hydrophilic proteins and positive values indicate hydrophobic proteins. The GRAVY index values range from −2 to +2. The Boman index, which is an estimate of the protein-binding potential, varies from 1.41 in DEFL4-8 82-94 to 4.21 in DEFL1-23 65-82 . A high Boman index value (more than 2.5) indicates that an AMP can interact with a wide range of proteins, and thus, is multifunctional. Most γ-core peptides have the Boman index values exceeding 2.5. Only in six peptides are the Boman index values lower ( Table 1). The hydrophobic moments (µH), which reflect the amphipathicity of an α-helix, were computed. The highest hydrophobic moment of 0.534 was in TkThi1 96-109 . Twelve peptides were predicted to be antimicrobial: all γ-core motif peptides designed from the sequences of classical DEFLs, one peptide derived from 4-Cys-containing DEFLs DEFL4-20 86-110 that spanned the 4-Cys motif, Tk-AMP-K2 10-23 , TkLTPd5.6 59-75 and TkSN1 39-57 .

3D Structure Simulation
The 3D structure of the γ-core peptides and their variants was predicted using PEP-FOLD3 ( Figure 2) [25]. All γ-core peptides of the classical defensins possess an α-helical region of different lengths (from 6 to 13 amino acid residues) and unstructured N-and Cterminal «tails». No β-structure was predicted in either peptide. The shortest α-helices of six residues were observed in DEFL1-16 65-82 and DEFL1-32 55-68 . In DEFL1-16 65-82 , the α-helix encompasses the conserved hexapeptide RGFRRR. In DEFL1-32 55-68 , the α-helix covers the sequence KFRRCT. The longest α-helix of 13 residues was predicted in DEFL1-20 65-82 .   The structure of the γ-core motif peptides of the 4-Cys-containing DEFLs is more diverse (Figure 2). The α-helices were predicted only in three peptides: DEFL4-20 92-102 , HvDEFL4-1 67-77 and DEFL4-20  . The latter has two α-helical regions: in the N-and Cterminal regions of the molecule, while the central part of the peptide is unstructured. The α-helices are short and consist of four amino acid residues. In DEFL4-20 86-110 , the second α-helix is longer and contains six amino acid residues. The γ-core motif peptides from the other 4-Cys-containing DEFLs were predicted to adopt the random coil conformation.
In the 3D structure of TkThi1 96-109 , a helical region was also predicted (residues G2−S13). In contrast, the Tk-AMP-K2 10-23 was predicted to be in a random coil conformation.

Antimicrobial Activity of Synthetic Peptides
The antimicrobial activity of synthetic peptides was tested against a panel of human and plant pathogens. Among them were the yeasts Candida albicans VKM Y-2994 and

Peptide Fragments of 4-Cys-Containing DEFLs
Studies of the antimicrobial activity of seven peptide fragments of 4-Cys-containing DEFLs showed that all of them suppressed the growth of yeasts, however, the efficiency of inhibition varied depending on the peptide tested ( Figure 3B). Usually, the activity of the longer peptides was higher than that of the truncated variants: DEFL4-20 86-110 was more potent than DEFL4-20 92-102 , DEFL4-37 90-102 was more efficient than DEFL4-37 91-101 , and DEFL4-8 82-94 was more effective than DEFL4-8 83-93 against Cr. neoformans. The opposite trend was observed for C. albicans. The peptides DEFL4-20 86-110 and DEFL4-37 90-102 had the highest activity against Cr. neoformans (95 and 100% inhibition, respectively). The efficiency of inhibition of Cr. neoformans by DEFL4-4 46-57 and C. albicans by DEFL4-37 90-102 was also rather high ( Figure 3B). The efficiency of inhibition of yeasts by other peptide fragments was lower (equal or below 26%). Of the pathogenic bacteria, Cl. michiganensis was suppressed by four peptides, while Ps. savastanoi and P. carotovorum were suppressed by five. The peptide DEFL4-20 86-110 exhibited the highest activity against Cl. michiganensis (91% inhibition). DEFL4-37 90-102 showed the highest activity against Ps. savastanoi. P. carotovorum was inhibited by 42 and 36% by DEFL4-8 82-94 and DEFL4-37 90-102 , respectively. Again, the longer peptides were more effective than the truncated derivatives. Studies of the antifungal activity of the fragments of the 4-Cys-containing DEFLs showed that F. oxysporum growth was suppressed by all seven peptides, F. verticillioides by six peptides, and F. culmorum and F. solani by four peptides. The highest activity (65% inhibition) was displayed by DEFL4-20 86-110 against F. culmorum. The same peptide was the most potent against F. oxysporum (52% inhibition). In contrast to the activity against yeasts and bacteria, no positive correlation was observed between the length of the peptide fragment and its activity against all Fusarium species. While DEFL4-20 86-110 was much more active than DEFL4-20 92-102 against F. culmorum and F. oxysporum, the truncated peptide DEFL4-20 92-102 was more potent against F. solani. The peptide DEFL4-37 90-102 was more effective than DEFL4-37 91-101 in inhibiting the growth of F. oxysporum and F. verticillioides; the activity of DEFL4-8 82-94 against F. oxysporum and F. solani was lower than that of DEFL4-8 83-93 .
The barley peptide HvDEFL4-1 67-77 showed the narrowest activity spectrum ( Figure 3B). Of the nine tested pathogens, it was active against four. It showed complete inhibition of Cr. neoformans, while C. albicans was suppressed by 25%. Inhibition of F. culmorum and F. verticillioides was 34 and 32%, respectively. Other pathogens were insensitive to HvDEFL4-1 67-77 .

Peptide Fragments of Other Wheat AMPs
The peptide fragment Tk-AMP-K2 10-23 , with a high efficiency, suppressed the growth of all the tested pathogens ( Figure 3C). Similar to other peptides, its activity was pathogendependent. The most sensitive pathogen to the peptide was Cr. neoformans (100% inhibition) followed by the Gram-negative bacteria Ps. savastanoi (80% inhibition) and P. carotovorum (70% inhibition). The efficiency of inhibition of the other pathogens, such as C. albicans, F. culmorum and F. verticillioides was also rather high (49, 42 and 35%, respectively). The degree of inhibition of two other Fusarium species (F. oxysporum and F. solani) as well as the Gram-positive bacterium Cl. michiganensis was much lower (11, 13 and 24%, respectively).
The snakin fragment TkSN1 39-57 was a very effective inhibitor of all tested microbes except F. verticillioides. The efficiency of inhibition varied from 18% (Ps. savostanoi) to 98% (C. albicans). Both the yeast species and Cl. michiganensis were the most sensitive pathogens, which were virtually completely inhibited by TkSN1 39-57 ( Figure 3C).

Dynamics of Pathogen Inhibition by γ-Core Motif Peptides
The dynamics of pathogen inhibition by γ-core peptides was studied, and the IC 50 values were calculated ( Figure 4, Table 2). The efficiency of inhibition by DEFL1-12 67-77 was the highest for Cr. neoformans and Cl. michiganensis and amounted to nearly a 100% inhibition of Cr. neoformans at the peptide concentration of 100 µM. The efficiency of inhibition of Cl. michiganensis reached 90% at the highest tested concentration of 300 µM. The peptide DEFL1-16 65-82 was much more active against most tested pathogens, especially against Cr. neoformans, C. albicans, Cl. michiganensis, and two Fusarium species: F. oxysporum and F. culmorum. The IC 50 values for these pathogens were in the range from 4.4 to 20.7 µM. The peptide DEFL1-20 65-82 inhibited the growth of F. oxysporum and F. culmorum, however, inhibition did not exceed 60%. The peptide DEFL1-23 65-82 was highly active against the yeast Cr. neoformans and the bacterium Cl. michiganensis. The inhibition of Cr. neoformans reached 98% at a concentration of 100 µM. F. culmorum and F. oxysporum were also highly susceptible to the peptide. The suppression of the growth of the Fusarium fungi amounted to 75% at the peptide concentration of 150 µM. The peptide DEFL1-32 55-68 was highly active against five pathogens. At the peptide concentration of 80 µM, Cr. neoformans was inhibited by 100%. C. albicans was suppressed by 92% at the highest tested concentration. The inhibition efficiency of three other pathogens (Cl. michiganensis, F. oxysporum and F. culmorum) reached 70−79% depending on the pathogen species. The peptide DEFL1-36 65-82 had a very broad host range, suppressing the growth of seven pathogens with the complete inhibition of Cr. neoformans and Cl. michiganensis at 50 and 100 µM, respectively. Inhibition of F. culmorum and F. oxysporum reached 80% at 50 and 70 µM, respectively. The maximum inhibition of other Fusarium species was in the range from 45 to 55%. The peptide DEFL1-40 65-82 was highly active against Cr. neoformans, Cl. michiganensis, F. oxysporum and F. culmorum. Almost a complete inhibition of 98% for Cr. neoformans was achieved at the peptide concentration of 100 µM. Cl. michiganensis was by 88% inhibited by the highest peptide concentration tested. A maximum inhibition of 78% was achieved for the two Fusarium species at the peptide concentration of 150 µM. Cr. neoformans was achieved at the peptide concentration of 100 μM. Cl. michiganensis was by 88% inhibited by the highest peptide concentration tested. A maximum inhibition of 78% was achieved for the two Fusarium species at the peptide concentration of 150 μM.
The peptide DEFL4-8 82-94 inhibited the growth of F. verticillioides and P. carotovorum. A maximum inhibition of 26 and 42%, respectively, was observed at the highest tested concentration. The peptide DEFL4-20 86-110 suppressed the growth of four pathogenic microbes. Cr. neoformans and Cl. michiganensis were suppressed by 95 and 91%, respectively, at the highest peptide concentration, while F. culmorum and F. oxysporum were suppressed by 60 and 54% at 150 µM. The peptide DEFL4-37 90-102 induced 100% inhibition of Cr. neoformans at the highest tested concentration. The maximum inhibition of F. verticillioides and C. albicans was about 35% at 150 and 300 µM, respectively.

Discussion
The survival of the growing human population worldwide depends on agricultural production. Among phytopathogens, fungi are the key causative agents of destructive crop plant epidemics; furthermore, they cause considerable and persistent losses of crop yields annually [26]. Plant diseases caused by fungi alone usually reduce by as much as a 30 percent of the crop harvest in the absence of appropriate and efficient management [27]. Bacterial diseases, which are widespread on all kinds of cultivated and commercial value plants and are especially difficult to identify and control, are also of great economic concern. The control of fungal and bacterial diseases is mainly based on the application of chemical fungicides. Only few pesticides including antibiotics and heavy metals are available to control bacterial infections. Chemical fungicides inhibit pathogens but are toxic to beneficial microorganisms, human and animal health, and the environment. Moreover, the emergence of resistant strains makes plant diseases increasingly chal-

Discussion
The survival of the growing human population worldwide depends on agricultural production. Among phytopathogens, fungi are the key causative agents of destructive crop plant epidemics; furthermore, they cause considerable and persistent losses of crop yields annually [26]. Plant diseases caused by fungi alone usually reduce by as much as a 30 percent of the crop harvest in the absence of appropriate and efficient management [27]. Bacterial diseases, which are widespread on all kinds of cultivated and commercial value plants and are especially difficult to identify and control, are also of great economic concern. The control of fungal and bacterial diseases is mainly based on the application of chemical fungicides. Only few pesticides including antibiotics and heavy metals are available to control bacterial infections. Chemical fungicides inhibit pathogens but are toxic to beneficial microorganisms, human and animal health, and the environment. Moreover, the emergence of resistant strains makes plant diseases increasingly chal-

Discussion
The survival of the growing human population worldwide depends on agricultural production. Among phytopathogens, fungi are the key causative agents of destructive crop plant epidemics; furthermore, they cause considerable and persistent losses of crop yields annually [26]. Plant diseases caused by fungi alone usually reduce by as much as a 30 percent of the crop harvest in the absence of appropriate and efficient management [27]. Bacterial diseases, which are widespread on all kinds of cultivated and commercial value plants and are especially difficult to identify and control, are also of great economic concern. The control of fungal and bacterial diseases is mainly based on the application of chemical fungicides. Only few pesticides including antibiotics and heavy metals are available to control bacterial infections. Chemical fungicides inhibit pathogens but are toxic to beneficial microorganisms, human and animal health, and the environment. Moreover, the emergence of resistant strains makes plant diseases increasingly chal-

Discussion
The survival of the growing human population worldwide depends on agricultural production. Among phytopathogens, fungi are the key causative agents of destructive crop plant epidemics; furthermore, they cause considerable and persistent losses of crop yields annually [26]. Plant diseases caused by fungi alone usually reduce by as much as a 30 percent of the crop harvest in the absence of appropriate and efficient management [27]. Bacterial diseases, which are widespread on all kinds of cultivated and commercial value plants and are especially difficult to identify and control, are also of great economic concern. The control of fungal and bacterial diseases is mainly based on the application of chemical fungicides. Only few pesticides including antibiotics and heavy metals are available to control bacterial infections. Chemical fungicides inhibit pathogens but are toxic to beneficial microorganisms, human and animal health, and the environment. Moreover, the emergence of resistant strains makes plant diseases increasingly challenging to treat. In addition to reduced agricultural food production, fungi can infect and cause diseases in humans, which affect approximately 25% of the total population and increase the rate of morbidity and mortality among immunocompromised individuals [28].
Accordingly, the development of ecologically friendly and non-toxic strategies to control fungal and bacterial infections of plants and opportunistic fungal infections in humans has become a priority. These alternative control measures include the use of plant-derived natural antimicrobials and biological control agents.
The effective use in disease control of plant AMPs as natural antibiotics requires the knowledge of their antimicrobial determinants and mode of action. The efficient exploitation of the biocontrol agents in plant disease management is also dependent on the elucidation of their mechanism of action. Earlier, we demonstrated that the intracellular metabolites of the non-pathogenic strain FS-94 of F. sambucinum induced resistance to the pathogenic F. oxysporum strains F37 and F-137 in tomato and wheat plants, respectively. Resistance development was accompanied by the up-regulation of an array of AMP-like genes [19,21,29]. However, their exact biological role in the immune response remained unknown. Structure-function studies of the classical defensins showed that the antimicrobial activity is associated, at least in part, with their γ-core motifs [17,30]. In our studies of tomato CRPs, we showed that the γ-core motifs of the CRPs belonging to other peptide families, such as snakins, MEG (Maternally Expressed Gene) peptides, and nsLTPs, also possess antimicrobial activity [18]. However, it remained unclear as to whether the γ-cores of the so-called 4-Cys DEFLs, knottin-and thionin-like peptides display antimicrobial properties. In this work, we assayed the antimicrobial activity of the γ-core peptides from the wheat AMPs belonging to the classical defensins, 4-Cys DEFLs, snakins, nsLTPs, knottins and thionins, which were up-regulated by the pathogenic fungus F. oxysporum or F. sambucinum resistance inducers, against an array of human and plant pathogens. We showed that all the tested γ-core peptides exhibited antimicrobial properties. This important finding proves the role of this motif in antimicrobial properties of AMPs of different families. It also assigns antimicrobial functions to the peptides, whose role in defense has not been elucidated so far.

Discovery of Antimicrobial Activity in 4-Cys DEFLs and Conservation of the γ-Core Motifs
For the first time, we detected antimicrobial activity in the γ-core peptides of several 4-Cys-containing DEFLs which proves their direct role in defense as antimicrobial agents. Note that until now, only two families of 4-Cys-containing AMPs have been reported in plants-α-hairpinins and NCR (Nodule Cysteine-Rich) peptides. In the hairpinins, discovered in a number of species including maize, wheat, chickweed, buckwheat, macadamia and some others, the cysteine arrangement is as follows: C-X{3}-C-X{n}-C-X{3}-C [31]. The fold of the hairpinins resembles a hairpin composed of two α-helices connected by two disulphide bridges. Hairpinins exert either antifungal or proteinase inhibitory activity [31]. NCR peptides comprise a large family of highly diverse peptides with a conserved 4-or 6-Cys motif which are specifically expressed in legume nodules [32]. The cysteine motif in 4-Cys-containing NCR peptides conforms to the following signature: C-X-{5}-C-X{n}-C-X{4}-C. Their 3D structure includes either one short C-terminal antiparallel β-sheet and a short α-helix as in NCR044 or only the β-sheet without α-helix as in NCR169 [33,34]. NCR peptides were shown to be involved in nodule development and defense [35].
The cysteine signature in 4-Cys DEFLs of wheat and barley is different from those of α-hairpinins and NCR peptides, and their 3D structure as well as functions remains obscure. Three of the wheat DEFLs taken for the γ-core peptide synthesis showed a high sequence similarity to the uncharacterized proteins of wheat diploid relatives, and one DEFL displayed no significant homology to any of the protein sequences in the public databases ( Figures S1, S2 and S4). Interestingly, homologues of the studied wheat 4-Cys DEFLs differing in the degree of sequence similarity/identity were discovered in other cereals (Figures S1-S5). For example, DEFL4-8 82-94 of the hexaploid T. kiharae showed high sequence similarity and identity in the γ-core region with the predicted 4-Cys DEFLs from the closely related species, such as Ae. tauschii, Triticum turgidum L. and Triticum aestivum L. High sequence similarity, especially in the γ-core motif region, was observed in the predicted 4-Cys DEFLs from more distantly related species, such as Thinopyrum elongatum (Host) D. R. Dewey (syn. Elytrigia elongata), Oryza sp., Zizania palustris L., Eragrostis curvula (Schrad.) Nees, and Brachypodium distachyon (L.) P. Beauv. (Figure S2). The γ-core motif identical to DEFL4-20 86-110 was discovered in DEFL4-34, DEFL4-35 and T. elongatum TeDEFL4-20 ( Figure S3, Table S1). Closely related sequences were found in 4-Cys DEFLs from the wheat DEFL4-10, KQJ82122.1 from B. distachyon and XP_002447609.1 from Sorghum bicolor (L.) Moench. DEFL4-37 90-102 was discovered in KAE8796982.1 from H. vulgare, with minor modifications, in the predicted T. elongatum 4-Cys DEFL, KAF7033286.1 of T. aestium, XP_037418557.1 from Triticum durum Desf., XP_020190562.1 from Ae. tauschii and DEFL4-38 from T. kiharae ( Figure S4). Sequences related to DEFL4-4 46-57 were found in predicted DEFLs of several Triticum and Aegilops species, TeDEFL4-4 from T. elongatum, KQJ82122.1 from B. distachyon and XP_044973437.1 from H. vulgare ( Figure S1). The widespread distribution of 4-Cys DEFLs among cereals and the conservation of their γ-core sequences in evolution point to the important role of this peptide family in the plant immune system and the significance of the γ-core motif for the antimicrobial activity. It is worth noting that conservation of the γ-core sequences was observed not only in 4-Cys DEFLs, but in other AMP families: classical defensins, snakins, nsLTPs, thionins ( Figures S6-S17), in which family members with identical γ-core motifs were discovered. For example, the γ-core sequence of DEFL1-16 shown to exert potent antimicrobial properties was detected in DEFLs from different species of grasses including both cultivated and wild-growing ( Figure S8). Furthermore, this sequence was found in the defensins of plants beyond the grass family [23].

Discovery of Antimicrobial Activity in the Knottin-like Peptide Tk-AMP-K2 10-23 and the C-Terminal Prodomain of the Thionin-like Protein TkThi1 96-109
In addition to 4-Cys DEFLs, we revealed antimicrobial activity in the knottin-like peptide Tk-AMP-K2 10-23 , which was earlier isolated from T. kiharae seeds [22]. According to the cysteine signature similar to that in conotoxins, this peptide was assigned to knottin-like peptides. In this work, we confirmed the antimicrobial activity of this peptide.
We also assayed the antimicrobial potential of the γ-core motif of the thionin-like peptide TkThi1. This motif was discovered not in the mature peptide domain, but in the prodomain of the TkThi1 preproprotein. Note, that all thionins are synthesized as preproproteins that contain a signal peptide and an acidic C-terminal propeptide [36]. Within species, the C-terminal propeptides, especially the position of the six cysteine residues in the polypeptide chain, are highly conserved [37]. There is also homology, although less pronounced, between the acidic propeptides of the thionins of different plant species. We discovered sequence similarity between the γ-core motifs of the prodomains of the wheat thionin-like peptide TkThi1 and the Oryza sativa L. thionin-like proteins (EAZ01134.1, XP_015641946.1, etc.) ( Figure S16). The sequence conservatism of the C-terminal prodomain indicates the functional importance of this precursor region, although its exact role has not been clarified so far. The C-terminal prodomain is thought to be essential for the targeted transport of the mature thionin to the vacuoles, cell walls or protein bodies. In addition, it is believed to neutralize the toxic properties of the mature peptide before it enters the intercellular space or the vacuole or acts as a chaperone that ensures thionin folding [38]. Our results show that TkThi1 96-109 exhibits antimicrobial activity, with the highest activity against the Gram-positive bacterium Cl. michiganensis (40% inhibition at the highest tested concentration). Therefore, previously unknown antimicrobial functions of the C-terminal thionin prodomain have been disclosed.

Antimicrobial Potency and Activity Spectrum of the γ-Core Peptides
In addition to revealing antimicrobial properties in CRPs with previously unknown functions, we also compared the antimicrobial potency and activity spectrum of all synthetic γ-core peptides to infer the problem of structure-function relationships and identify the peptides most suitable for control of particular pathogens.
Antimicrobial assays of synthetic γ-core peptides revealed that all of them were active against several pathogens. However, the degree of inhibition as well as the activity spectrum varied depending on the peptide and the pathogen species. This might result from the different physicochemical characteristics of the tested synthetic peptides.
The activity spectrum was the broadest for DEFL1-16 65-82 and Tk-AMP-K2 10-23 : both peptides inhibited all nine tested pathogens ( Figure 3). However, the degree of inhibition of most pathogens was higher for DEFL1-16 65-82 . Six peptides were active against eight of nine pathogens. Of them, DEFL1-36 65-82 and TkSN1 39-57 had the highest activity against the sensitive microorganisms. The high inhibitory activity of DEFL1-16 65-82 , DEFL1-36 65-82 and TkSN1 39-57 against most tested pathogens correlates with the high net positive charge of their molecules (+8 and +7, +5, respectively). Conversely, the low charge of the molecules (+1) correlates with the low antimicrobial potency of DEFL4-8 83-93 , DEFL4-8 82-94 , DEFL4-20 92-102 and TkThi1 96-109 . The importance of the high charge for the antimicrobial activity of α-helical peptides was shown by Zelezetsky and Tossi [39]. On the model peptides it was demonstrated that decreasing the charge below +3 reduced antimicrobial potency [39]. It was also shown that the activity depended on the overall cationicity and not necessarily on the location of positively charged residues in the polypeptide chain [39]. On the whole, the tested γ-core peptides with the net charge of +1 were less active than those with the higher net charge. However, besides the charge, other factors also contributed to the antimicrobial activity of the γ-core peptides. This follows from the observation that the peptides with an identical charge differed in antimicrobial properties. The importance of the amphiphilic structure for potent, broad-spectrum activity was demonstrated for the α-helical AMPs [39]. However, our experiments show that the peptide TkThi1 96-109 with the highest hydrophobic moment of 0.534 and predicted to be α-helical exhibited moderate and narrow-range activity when compared to other peptides. The role of hydrophobicity in the antimicrobial activity of α-helical peptides was also repeatedly emphasized. Hydrophobicity is assumed to be significant for penetration of the peptide into the lipid bilayer of the membranes of pathogens. In our assays, the ratio of hydrophobic residues in one of the most potent peptides DEFL1-16 65-82 as well as its hydrophobic moment was rather low when compared to much more hydrophobic and amphiphilic but less-active peptides, such as TkThi1 96-109 and Tk-AMP-K2 [10][11][12][13][14][15][16][17][18][19][20][21][22][23] . In summary, our results show that any, even subtle changes in the physicochemical properties of peptides can have a pronounced effect on the potency and spectrum of antimicrobial activity.

Structure-Function Relationships
The comparison of antimicrobial properties of the γ-core motif peptides with high sequence similarity allowed us to draw conclusions on the role of certain amino acid residues in the antimicrobial activity. Thus, by analyzing the antimicrobial activity of DEFLderived peptides, we discovered that DEFL1-32 55-68 was among the most potent peptides. Conversely, DEFL1-11 55-68 was among the least active peptides. However, DEFL1-11 55-68 differs from DEFL1-32 55-68 only by two amino acid residues: a dipeptide IS in DEFL1-11 55-68 is substituted by FR in DEFL1-32 55-68 . This substitution increases the charge of DEFL1-32 (Table 1), affects its 3D structure and dramatically enhances its antimicrobial potency (Figures 3 and 4). Accordingly, the dipeptide FR in the γ-core sequence of DEFL1-32 is vital for its antimicrobial activity.
A comparison of the antimicrobial properties of a pair DEFL1-23 65-82 /DEFL1-40 65-82 , which differ by a single aromatic residue (Y13F) between the second and the third cysteines, shows that the activity spectrum of both peptides is very similar, as well as the antimicrobial potency against the tested microbes, pointing to the minor role of Y versus F in the antimicrobial activity of these peptides. Sequence variation in another pair, DEFL1-20 65-82 /DEFL1-23 65-82 , which consists of a single non-conserved substitution of L7 in DEFL1-20 65-82 for R7 in DEFL1-23 65-82 resulting in a higher positive charge of DEFL1-23 65-82 , had a more pronounced effect on the degree of pathogen inhibition: DEFL1-20 65-82 was unable to suppress the growth of Ps. savastanoi, while DEFL1-23 65-82 inhibited this pathogen by 30% (Figure 3). DEFL1-23 65-82 was also more active than DEFL1-20 65-82 against F. culmorum, F. oxysporum, and Cr. neoformans indicating the importance of R7 in the γ-core motif for the antimicrobial activity against these pathogens. At the same time, DEFL1-20 65-82 inhibited C. albicans by 14%, while DEFL1-23 65-82 was ineffective against this pathogen.
Our results show that the charge of the peptide is not the only determinant of antimicrobial activity. This follows from the comparison of the antimicrobial properties of the pair DEFL1-20 65-82 /DEFL1-32 55-68 . The peptides have the same net charge; however, their sequences vary, which correlates with differences in antimicrobial activity against particular pathogens. In contrast to DEFL1-20 65-82 , DEFL1-32 55-68 is active against Ps. savastanoi and highly active against Cr. neoformans and C. albicans, while DEFL1-20 65-82 shows better activity against Cl. michiganensis. This result shows that besides the charge of the peptide, its amino acid sequence is essential for the antimicrobial activity. The same holds true for the pair TkLTP2.25 50-62 /TkLTPd5.6 59-75 . Both TkLTP2.25 50-62 and TkLTPd5.6 59-75 have the same net charge of +3 at pH 7.0. However, the antimicrobial activity of both peptides differs considerably: TkLTP2.25 50-62 was much more active against the majority of tested pathogens. This means that the sequence of the nsLTP-derived peptides is likely to influence the antimicrobial activity despite the overall similarity in the 3D structure.

Mode of Action
Studies of the mode of action of the eight most active γ-core motif peptides from the AMPs of different families (DEFLs, snakins, nsLTPs, knottins) on yeast cells using staining with propidium iodide showed that all the tested peptides induced membrane permeabilization (Figures 5-7). For three peptides (DEFL4-37 90-102 , HvDEFL4-1 67-77 and Tk-AMP-K2 10-23 ), flow cytometry showed a concomitant reduction in the number of Cr. neoformans cells, pointing to the peptide-induced cell lysis. Accordingly, the tested peptides disturbed the membrane integrity, which could be the cause of the pathogen growth arrest and cell lysis, either alone or in combination with some other mechanism, e.g., inhibition of DNA or protein synthesis, etc. We can hypothesize that the cationic nature of the peptides provided an initial interaction with the negatively charged phospholipids of the plasma membranes, followed by the formation of pores or membrane disruption by some other mechanism resulting in leakage of the cell constituents. Inhibition dynamics studies of DEFL1-32 55-68 against C. albicans and DEFL4-37 90-102, HvDEFL4-1 67-77 and Tk-AMP-K2 10-23 against Cr. neoformans demonstrated a low inhibition level until a certain critical peptide concentration was achieved, resulting in near-complete inhibition of the pathogen (Figure 4). We speculate that such inhibition dynamics are consistent with the carpet model of membrane disintegration by the membrane-active peptides. In the carpet model, the peptides accumulate in parallel on the surface of the membrane. At a threshold concentration of peptides, the displacement of the phospholipids occurs with subsequent membrane disruption [40].

Chemical Synthesis of Peptide Fragments Derived from AMP-like Peptides
Peptide fragments of wheat and barley AMP-like peptides were produced by solidphase synthesis using Fmoc chemistry (Elabscience Biotechnology Inc., Wuhan, China). The synthesized peptides were purified by RP-HPLC. Their identity to the required sequences was proved by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis on an Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in a linear or reflector positive ion mode using α-cyano-4hydroxycinnamic acid as a matrix.
The following characteristics of the synthesized peptides were calculated using the ExPASy ProtParam tool [41]: molecular weight, pI, net charge at pH 7, GRAVY index and aliphatic index. Hydrophobic moment µH was calculated with HeliQuest [42]. The Boman index was computed using APD3 [43]. The prediction of antimicrobial properties was carried out with CAMPR3 [44].

3D Structure Modeling
The spatial structure of the synthesized peptides was de novo modeled using the PEP-FOLD3 program [25]. The best representative models were those with the lowest sOPEP values provided by PEP-FOLD3.

Antimicrobial Assays
The antimicrobial activity of synthetic peptides was tested against the yeasts Candida albicans VKM Y-2994 and Cryptococcus neoformans VKM Y-2755, the bacteria Pseudomonas savastanoi pv. savastanoi VKM B-1546, Pectobacterium carotovorum subsp. carotovorum VKM B-1247 and Clavibacter michiganensis subsp. michiganensis VKM Ac-1403, and four Fusarium species: F. culmorum VKM F-2303, F. oxysporum VKM F-137, F. solani VKM F-142 and F. verticillioides VKM F-670. All cultures were obtained from the All-Russian Collection of Microorganisms (VKM). The details of the production and storage of cultures of microorganisms used in this study are described at the VKM Home page [45]. Antimicrobial assays were accomplished according to the procedure described earlier [18]. In short, yeasts were grown on the YPD-P medium and the bacteria P. carotovorum and Cl. michiganensis were cultured on a modified YPD-P medium [18]. The Fusarium fungi were grown on potato dextrose agar at 25 • C for 7-8 days, and spores were washed off from the surface of the mycelia with sterile, distilled water. The antimicrobial activity of the peptides was measured in immunoassay microtiter plates according to the protocol of Broekaert et al. [46]. With yeasts and bacteria, each well contained 10 µL of the tested peptide solution (final concentrations of 10-300 µM) in water, 80 µL of the medium and 10 µL of the microbial suspension. The plates were incubated at 30 • C for 24 h, and the absorbance of the suspension was measured on an Efos 9305 spectrophotometer (Sapphire, Moscow, Russia) at 594 nm. For Fusarium species, the wells of a microtiter plate were filled with 90 µL of the fungal spore suspension in half-strength potato dextrose broth at a concentration of 2000-3000 spores in 100 µL and 10 µL of aqueous solutions of peptides at final concentrations of 10-300 µM. After 38 h of incubation, the absorbance was recorded at 595 nm on a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Antimicrobial activity is expressed in IC 50 values, which show the concentration necessary for 50% inhibition of the pathogen growth; this was determined from a graph showing the activity dependence on peptide concentration.

Statistical Analysis
For each pathogen, experiments on the peptide antimicrobial activity evaluation were carried out in triplicate per treatment. Mean values, standard deviations (SD), and the significance of differences (p ≤ 0.05) of the means between treatments and controls (t-test for independent variables) were determined using STATISTICA v. 6.1 software (StatSoft Inc., Tulsa, OK, USA).

Staining of Yeast Cells with Propidium Iodide
Staining of yeast cells with propidium iodide in the presence of the γ-core peptides was carried out according to the protocol described earlier [18]. C. albicans or Cr. neoformans cells (24 h culture in YPD-P at a cell concentration of 2 × 10 8 /mL) were incubated with the peptide at a concentration of 300 µM at 30 • C for 1 h. After incubation, 1 mL of the cells was stained with 0.03 mM propidium iodide (Sigma, St. Louis, MO, USA). The staining proceeded for 15 min at 30 • C. The fluorescence was recorded on an AXIO Imager A1 fluorescence microscope (ZEISS, Göttingen, Germany) using a Zeiss filter set 56 HE.
For flow cytometry experiments with Cr. neoformans, the yeasts were grown on the YPD medium at 28 • C for 48 h. After that, the cells were centrifuged at 12,000× g for 3 min, washed with MiliQ water and incubated with the peptides dissolved in water at a concentration of 300 µM at 28 • C for 2 h. The cells were stained with propidium iodide by direct addition of the dye to the incubation medium (final concentration of 2 µg/mL) and measured on a NovoCyte Flow cytometer (Agilent, Santa Clara, CA, USA) using the channel FL2 (488 nm excitation and 585 nm emission). The sample without peptides was used as a control.

Conclusions
In this work, we report the antimicrobial activity of 21 γ-core peptides of 18 AMPs of wheat T. kiharae and barley H. vulgare against an array of plant and human pathogens. All the tested peptides displayed antimicrobial properties which depended on the pathogen species. Conservation of the γ-core motif regions in AMPs of the grass species was revealed. Peptides especially efficient against particular pathogens, which may find practical application for design of novel antimicrobials, were revealed. The discovery of antimicrobial activity in 4-Cys DEFLs, the knottin-like peptide Tk-AMP-K2 and the C-terminal prodomain of the thionin-like protein TkThi1 points to their direct role in defense mechanisms. Studies of the mode of action of the eight most potent peptides against yeast cells show that they trigger membrane permeabilization resulting in cell lysis. Whether the peptides interact with intracellular targets is still to be explored.

Conflicts of Interest:
The authors declare no conflict of interest.