Research Article

Received: 4 July 2010 Revised: 28 November 2010 Accepted: 25 January 2011 Published online in Wiley Online Library: 28 March 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4359

A novel antifungal peptide from foxtail millet seeds

Wentao Xu,a,b Lu Wei,a Wei Qu,a Zhihong Liang,a Jinai Wang,a Xiaoli Peng,b Yanan Zhangb and Kunlun Huanga,b
Abstract
BACKGROUND: Antifungal proteins (AFP) help plants to combat phytopathogenic fungi and thus protect plants from the devastating damage caused by fungal infections and prevent massive economic losses. To date, several proteins with antibacterial and/or antifungal properties have been isolated and characterized from different plant species and tissues; however, there are no reports concerning the antifungal peptide from foxtail millet seeds. RESULTS: An antifungal peptide with a molecular mass of 26.9 kDa was isolated from dry seeds of the foxtail millet (Setaria italica (L.) Beauv.), using a procedure that involved four chromatographic steps. The antifungal peptide was adsorbed on CM-Sepharose, Af-gel blue gel and Superdex 75. It was further puried by C18 reverse-phase high-performance liquid chromatography and submitted for analysis of peptide mass ngerprint. The Mascot peptide mass ngerprint of the isolated protein hit no existing protein (score >60), and it was proved to be a novel antifungal peptide. It inhibited mycelial growth in Alternaria alternate with an IC50 of 1.3 mol L1 , and it also exhibited antifungal activity against Trichoderma viride, Botrytis cinerea and Fusarium oxysporum. Transmission electron microscopy of mold forms of Alternaria alternate after incubation with 20 g mL1 of the antifungal protein for 48 h revealed marked ultrastructural changes in the fungus. CONCLUSION: A novel antifungal peptide with high potency was isolated from foxtail millet seeds. c 2011 Society of Chemical Industry Keywords: foxtail millet; antifungal peptide; isolation; peptide mass ngerprint; transmission electron microscopy

INTRODUCTION
Antifungal proteins (AFP) help plants to combat phytopathogenic fungi and thus protect plants from the devastating damage caused by fungal infections and prevent massive economic losses. Systemic production of the antifungal proteins in crop plants should be taken into account as a novel strategy tos engineer biological control of fungal pathogens, as shown for AFP and other antimicrobial proteins.1 4 Finally, they could complement or substitute for chemical preservatives in food, as suggested for Sodium uoride (molecular formula is NaF).5 Once the active sites of these proteins are determined, the activity and specicity range could be enhanced by site-directed mutagenesis and/or by the development of synthetic derivatives, in order to design new compounds with pharmaceutical value.6 8 Consequently, research on antifungal proteins has attracted the attention of many investigators. Foxtail millet (Setaria italica (L.) Beauv.) is an important food crop gown in India, China and Japan. It is also planted in Australia, North Africa and South America.9 It has long been favored by many local farmers for its excellent drought resistance, high tolerance to poor soil and good nutrient value. It has become increasingly popular and has formed a common dish for many families in recent years because of its nutritive value.10,11 Minor millets are nutritionally superior to rice and wheat and the presence of all the required nutrients in millets makes them suitable for

industrial-scale utilization in the manufacture of foodstuffs (e.g. baby foods, snack foods and dietary food).12,13 However, the productivity of foxtail millet is limited for various reasons, one of which is its susceptibility to fungal diseases such as downy mildew and ergot, resulting in heavy losses of yield and quality. To date, several proteins with antibacterial and/or antifungal properties have been isolated and characterized from different plant species and tissues;14 40 however, there are no reports about the antifungal peptide from foxtail millet seeds. A detailed analysis of antifungal peptide that could be effective against these diseases will therefore be used as an important step towards the nal objective of enhancing the defense mechanism of foxtail millet. This work will enrich the antimicrobial peptide family and may broaden our insight into the plant defense mechanism.

Correspondence to: Kunlun Huang, Laboratory of Food safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. E-mail: hkl009@163.com

These three authors contributed equally. a Laboratory of Food safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China b Supervision and Testing Center of Agricultural Products Quality, Ministry of Agriculture, Beijing 100083, China

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Figure 1. Ion-exchange chromatography on CM-Sepharose. Column dimensions: 2.5 cm 25 cm. Sample: the protein was puried by the saturated ammonium sulfate method and ltered using a 0.45 m membrane lter. Starting buffer: 50 mmol L1 HAc-NaAc buffer (pH 4.6). Line b across the right half of the chromatogram represents 01 mol L1 NaCl concentration gradient used to desorb adsorbed proteins. Antifungal activity was detected in fraction CM3 (line a).

Figure 2. Afnity chromatography on an Af-gel blue gel column. Column dimensions: 5 cm 10 cm. Sample: fraction CM3 from CM-Sepharose. Starting buffer: 20 mmol L1 Tris-HCl buffer (pH 7.4). Line b across the right half of the chromatogram represents 01 mol L1 NaCl concentration gradient used to desorb adsorbed proteins. Antifungal activity was detected in fraction B3 (line a).

MATERIALS AND METHODS

Purication of antifungal peptide Fresh foxtail millet seeds (800 g) were extracted with distilled water using a Waring blender, followed by centrifugation at 12 000 g for 20 min. The resulting supernatant was saved. The proteins were fractionated with ammonium sulfate (AS) by stepwise precipitation, and the concentrations of saturated AS were selected as 3060%. The precipitation was dialyzed against distilled Water for 24 h at 4 C to remove salinity, and then the water fraction containing peptides was freeze-dried and stored at 20 C until needed. After homogenization in distilled water (3 mL g1 ) and centrifugation (12 000 g for

20 min), the resulting supernatant was loaded on to a 2.5 cm 25 cm column of CM-Sepharose (Sigma, St Louis, MO, USA) in 50 mmol L1 HAc-NaAc buffer (pH 4.6). Five peaks were obtained. Fraction CM3 was subjected to afnity chromatography on a 5 cm 10 cm column of Af-gel blue gel (Bio-Rad, Sunnyvale, CA, USA) in 20 mmol L1 Tris-HCl buffer (pH 7.4). The third adsorbed fraction (B3) was subjected to gel ltration on a fast performance liquid chromatography (PLC) Superdex 75 HR 10/30 column (Amersham Biosciences, Little Chalfont, UK) in 50 mmol L1 sodium phosphate buffer (pH 7.2) containing 150 mmol L1 NaCl using an AKTA Purier (Amersham Biosciences). The rst fraction (SU1) represented puried antifungal peptide. High-performance

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of its electrophoretic mobility with those of molecular mass marker proteins from Amersham Biosciences. Matrix-assisted laser desorption ionization time-of-ight mass spectrometry (MALDITOF MS) was also employed to determine the molecular mass of the puried peptide (autoex II TOF/TOF, Bruker-Daltonics, Billerica, MA, USA). Protein microsequencing and peptide mass Fingerprinting The puried antifungal was separated by SDS-PAGE and electroeluted to a polyvinylidene diuoride (PVDF) membrane. It was visualized with Coomassie Blue and submitted to a commercial institution for N-terminal sequence assay by Edman degradation with an Applied Biosystems sequencer (model Procise 491). For peptide mass ngerprinting, isolation of the protein was performed by electrophoresis. The target protein spots were excised from the gel directly and then subjected to in-gel trypsin digestion.37 Mass spectrometry was carried out at the College of Biological Sciences of China Agricultural University on a MALDI-TOF MS (autoex II TOF/TOF, Bruker-Daltonics). The obtained peak lists were submitted to Matrix Sciences (http://www.matrixscience.com) programmed for protein identication. Additional match of the mascot peptide mass ngerprint of the foxtail millet seeds antifungal peptide with the other protein sequence in http://www.ncbi.nlm.nih.gov/blast was also performed (http://www.expasy.org/tools/ndpept.html). Antifungal activity assays The antifungal potency of the peptide was examined in different species of fungi, including Trichoderma viride, Alternaria alternate, Botrytis cinerea and Fusarium oxysporum. It was assessed by using sterile Petri plates (100 mm 15 mm) containing 10 mL potato dextrose agar. After the mycelial colony had developed, sterile Oxford cups were placed at a distance of 0.5 cm from the rim of the mycelial colony. An aseptic solution of the tested sample was prepared in 50 mmol L1 sodium phosphate buffer (pH 7.2). An aliquot of a solution of the isolated peptide was added to an Oxford cup. The Petri plates were incubated at 26 C for 72 h until

Figure 3. Gel ltration by fast protein liquid chromatography on a Superdex 75 HR 10/30 column using an AKTA purier. Sample: fraction B3 from Af-gel blue gel column. Buffer: 50 mmol L1 sodium phosphate buffer (pH 7.2). Flow rate: 0.5 mL min1 . Fraction size: 0.5 mL. Line b represents 0.15 mol L1 NaCl concentration. Antifungal activity was detected in fraction SU1 (line a).

liquid chromatography (HPLC) with a Kromasil 100-5 C18 column (4.6 mm 250 mm, 5 m) was used to further purify the antifungal compounds (mobile phase: acetonitrilewater = 90/10; ow rate 1 mL min1 ; temperature 25 C; total retention time 30 min). The elution was monitored at 220 nm. In order to obtain pure peptides in quantities sufcient for analysis, several runs were performed and the active fraction was collected. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), molecular mass determination SDS-PAGE was conducted according to the method of Laemmli and Favre36 with 5% stacking gel and 15% resolving gel using a vertical electrophoresis system (Bio-Rad). After electrophoresis the gel was stained with Coomassie Brilliant Blue. The molecular mass of the antifungal peptide was determined by comparison

Figure 4. HPLC with a Kromasil 100-5 C18 column (4.6 mm 250 mm, 5 m). Mobile phase: acetonitrilewater, 90/10; ow rate, 1 mL min1 ; temperature, 25 C; total retention time, 30 min. The elution was monitored at 220 nm.

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A novel antifungal peptide mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around the disks with antifungal samples.38 40 To determine the IC50 value for the antifungal activity, three doses of the antifungal peptide of foxtail millet seeds were added separately to three aliquots each containing 10 mL potato dextrose agar at 45 C, mixed rapidly and poured into three separate small Petri dishes. After the agar had cooled, a small drop of the mycelia suspension was placed in the center of each dish and then incubated at 26 C for 72 h. When the mycelia of the blank grew and arrived at the edge of the plate, the diameter of the mycelia area in each dish was measured. From the average diameter of three repeated determinations, the doses in g mL1 of 50% inhibition of mycelia extension (IC50 ) were obtained.39,41 43 Effect of antifungal peptide on hyphal morphology To evaluate the effect of the antifungal peptide on fungal hyphae, we proposed an application using coverslips. Fungi were grown on 100 mm 15 mm Petri plates containing 10 mL potato dextrose agar (PDA) and incubated at 26 C for 72 h. After some Oxford cups were placed at a distance of 0.5 cm from the rim of the mycelial colony, blank control and the isolated protein were added and then coverslips were inserted into the PDA along the direction of the hyphae. After 48 h, hyphal growth and morphology were examined and photographed with a light microscope (Olympus BX51T-DP70, Shinjuku-Ku, Tokyo, Japan) at 600 to examine structural abnormalities. Transmission electron microscopy In order to investigate the effect of foxtail millet seed antifungal peptide on the ultrastructure of Alternaria alternate forms, fungal samples were examined by transmission electron microscopy. Alternaria alternate treated with the isolated protein (subinhibitory concentration) was xed with 2.5% glutaraldehyde in 0.1 mol L1 cacodylate buffer (pH 7.2). Subsequently, they were washed in

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Table 1. Yields of chromatographic fractions (from 800 g seeds) Fraction Crude extract Ammonium sulfate precipitation (3060%) CM3 B3 SU1 HPLC Yield (mg) 3640 2973 Recovery (%) 100 81.7 Purication (-fold) 1

222.4 65.3 27.5 26.7

7.5 29.4 42.1 97.1

3.7 11.4 42.2 42.7

cacodylate buffer and postxed in 1% (w/v) OsO4 in 0.1 mol L1 cacodylate buffer (pH 7.2) with 1% potassium ferrocyanide and 5 mmol L1 CaCl2 for 30 min at room temperature. The postxed cells were dehydrated in acetone and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a transmission electron microscope (JEM-1230, JEOL, Tokyo, Japan).

RESULTS
The yields of the various chromatographic fractions are presented in Table 1. Antifungal activity was detected in the fraction of foxtail millet seeds extract adsorbed on CM-Sepharose (CM3) (Fig. 1) and subsequently adsorbed on Af-gel blue gel (B3) (Fig. 2). Fraction B3 was fractionated on Superdex 75 into two fractions. Antifungal activity resided in the rst fraction (SU1) (Fig. 3). The active fractions were puried in the nal step by reverse-phase chromatography on a C18 silica column (Fig. 4) and a single peak of antifungal activity was obtained. This peak appeared as a single band in SDS-PAGE (Fig. 5(a)). It represented puried antifungal protein.

Figure 5. Identication results of antifungal peptide of foxtail millet seeds by (a) SDS-PAGE and (b) mass spectrometry. (a) Left lane: molecular mass standard from Amersham Biosciences; right lane: puried antifungal peptide (fraction HPLC-1). (b) 100 pmol of the puried peptide was dissolved in watermethanol (50 : 50, v/v) containing 1% (v/v) acetic acid at a peptide concentration of 5 mol L1 . The determined molecular mass by spectrometry was 26898.842.

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Figure 6. Analysis of the antifungal peptide by MALDI-TOF MS.

Figure 8. Determination of IC50 value of antifungal activity of antifungal peptide of foxtail millet seeds toward Alternaria alternate. (A) Control; (B) 0.6 mol L1 antifungal protein; (C) 3 mol L1 antifungal protein; (D) 15 mol L1 antifungal protein. IC50 was determined to be 1.3 mol L1 .

The puried peptide yielded a single well-resolved peak on mass spectrometry and purity was about 96.8%, calculated using UNICORN software (Pharmacia & Biotech, Piscataway, New Jersey, USA). Its molecular mass was about 26.9 kDa (Fig. 5(b)). N-terminal sequence assay of the puried peptide was carried out by Edman degradation, but it could not be sequenced because of the NH2 terminal block. The antifungal peptide was analyzed further by MALDI-TOF MS (Fig. 6). The Mascot peptide mass ngerprint of this protein hit no existing protein (score >69, http://www.matrixscience.com), and the closest sequence similarity to hypothetical protein (Vitis vinifera, gi 147 865 223) (score, 59) suggested that this peptide may be a novel peptide. Additional match of the mascot peptide mass ngerprint of the foxtail millet seed antifungal peptide with the other protein sequence in http://www.ncbi.nlm.nih.gov/was also performed (http://www.expasy.org/tools/ndpept.html). The

highest sequence similarity ( mass, 0.005 Da) was found in defensin (Setaria italic) (ABM89231), but only 2.36% could be matched. Antifungal activity of the peptide toward the fungi Trichoderma viride and Alternaria alternate is illustrated in Fig. 7(a) and (b), respectively. Its antifungal activity toward Alternaria alternate with IC50 was determined to be 1.3 mol L1 (Fig. 8). Analysis of hyphal growth and morphology of Alternaria alternate is presented in Fig. 9. The morphological toxicity of our isolated protein to Alternaria alternate was revealed in mycelial apex offshoot, distortion, tumescence and rupture after treatment with 20 mol L1 at 26 C for 72 h. The effect of the antifungal protein on the ultrastructure of Alternaria alternate is presented in Fig. 10. Analysis of the images showed that control samples had fungal cells surrounded by a specic cell wall composed of dense outer and inner layers

Figure 7. Antifungal activity of puried antifungal peptide of foxtail millet seeds toward (a) Alternaria alternate and (b) Trichoderma viride. (a) (A) Control: 100 L of 50 mmol L1 PBS buffer (pH 7.2); (B) 20 mol L1 antifungal protein; (C) 2.5 mol L1 antifungal protein. (b) (A) Control: 15 L 50 mmol L1 PBS buffer (pH 7.2); (B) 50 mol L1 antifungal protein; (C) 30 mol L1 antifungal protein.

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Figure 9. Microphotographs of Alternaria alternate mycelia grown on PDA with or without antifungal peptide of foxtail millet seeds. (A) Control, normal mycelia of Alternaria alternate; structure is homogenous; (B) control, normal mycelial apex; (C) abnormal mycelia treated with antifungal peptide; (D) abnormal mycelial apex treated with antifungal peptide. The mycelial apex became distorted with the budding of mycelial apex and unusual structures clearly visible. In addition, some anomalies such as small swellings along the mycelia and mycelial apex were visible; (E)(H) abnormal mycelia and mycelial apex treated with antifungal peptide, showing mycelia with greater anomalous structures, budded mycelia apex, with cytoplasmic granulations, mycelia showing clear separation of cytoplasm from cell wall; (I)(L) abnormal mycelia treated with antifungal peptide, showing clear decrease in cytoplasmic content. The cytoplasmic reaction and mycelia were damaged.

Figure 10. Transmission electron microscopy of Alternaria alternate fungi forms. Normal ultrastructure of untreated fungi: delimited cell wall composed of dense outer and inner layers separated by a low-density space, and cell with a normal budding prole (A and B) in contrast to the altered morphology after treatment with 20 g mL1 of the antifungal peptide for 24 h at 37 C: deformed cells, alteration of the space between the cell wall and the plasma membrane (C, D, F), and irregular budding prole (E). Similar results were obtained in the different analyses.

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www.soci.org separated by a low-density space (Fig. 10(A, B)). Treated cells showed an undened and thickened cell wall, and a change in the space between the cell wall and the plasma membrane (Fig. 10(C, E, F)). The images showed completely deformed cells, with irregular budding sites (Fig. 10(D)).

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DISCUSSION
To purify the antifungal peptide, different concentrations of saturated ammonium sulfate were selected (30%, 40%, 50%, 60%, 70% and 80%). From the SDS-PAGE results and the inhibitory activity of antifungal peptide on mycelial growth in Alternaria alternate, we selected the best concentrations, which was the range from 30% to 60%. The antifungal peptide of foxtail millet seeds was adsorbed on CM-Sepharose, Af-gel blue gel and Superdex 75. The nal purication step was C18 reverse-phase HPLC. We found that the optimized purication procedure in this paper could more efciently gain the antifungal peptide of the foxtail millet seeds, but it was a little different from the purication procedure proven useful for the purication of other antifungal proteins and peptides.20 35,44 Although many studies have focused on showing the antifungal activity of plant derivatives, few have demonstrated their effects on the morphology and ultrastructure of the fungi. The data obtained in this study showed that Alternaria alternate underwent remarkable alterations, which were visible by light microscope and electron microscopy when treated with the antifungal peptide. The hyphae showed lack of cytoplasm, damage and loss of integrity and rigidity of the cell wall. The ultrastructural changes included thickening of the cell wall, an alteration of the space between the cell wall and the plasma membrane, deformed cells, reduction in cell size and cells with irregular budding sites. These observations indicated that the mode of antifungal activity of foxtail millet antifungal peptide was a result of its attack on the cell wall, retraction of cytoplasm in the hyphae and ultimately death of the mycelium. Such modications may be related to interference of the antifungal peptide components by enzymatic reactions of cell wall synthesis, affecting fungal morphogenesis and growth. The mechanism of action is not well understood; however, its deleterious effect on the cell wall of the fungus may be the main reason, because the integrity of the cell wall is necessary for cell division and to allow the expression of molecules involved in the adherence process. Further studies are needed to determine the nature and specic functions of this protein. In summary, an antifungal peptide with high potency was isolated from foxtail millet seeds.

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ACKNOWLEDGEMENTS
This work was supported by the Ministry of Agriculture and Ministry of Science and Technology of China.
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