A Buckwheat Antifungal Protein with Biocontrol Potential to Inhibit

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Bioactive Constituents, Metabolites, and Functions

A Buckwheat Antifungal Protein with Biocontrol Potential to Inhibit Fungal (Botrytis cinerea) Infection of Cherry Tomato Caicheng Wang, Susu Yuan, Weiwei Zhang, Tzi Bun Ng, and Xiujuan Ye J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01144 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Journal of Agricultural and Food Chemistry

1

A Buckwheat Antifungal Protein with Biocontrol Potential to Inhibit Fungal

2

(Botrytis cinerea) Infection of Cherry Tomato

3 4

Caicheng Wang,†,‡,§,‖ Susu Yuan,†,‡,§,‖ Weiwei Zhang,†,‡,§ Tzibun Ng,¶ Xiujuan Ye†,‡, §,*

5 6

†State

7

Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

8

‡Key

9


Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian

Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian

10

§Fujian

11


12

¶School

13

Kong, Shatin, Hong Kong, 999077, China

14

*

15

‖

Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian

of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong

Corresponding Author: Xiujuan Ye, [email protected] These

authors

have

contributed

equally

1

ACS Paragon Plus Environment

to

this

work


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ABSTRACT: A 11-kDa antifungal protein FEAP was purified from buckwheat

17

(Fagopyrum esculentum) seed extract with a procedure involving (NH4)2SO4

18

precipitation, and chromatography on SP-Sepharose, Affi-gel blue gel, Mono S and

19

Superdex peptide. Its N-terminal sequence was AQXGAQGGGAT resembling those

20

of buckwheat peptides Fα-AMP1 and Fα-AMP2. FEAP exhibited thermostability (20-

21

100 °C) and acid resistance (pH 1-7). Its antifungal activity was retained in presence of

22

10-150 mmol/L of K+, Mn2+ and Fe3+ ions, 10-50 mmol/L of Ca2+ and Mg2+ ions and

23

50% methanol, ethanol, isopropanol and chloroform. Its half-maximal inhibitory

24

concentrations toward spore germination and mycelial growth in Botrytis cinerea were

25

79.9 and 236.7 μg/mL, respectively. Its antifungal activity was superior to the fungicide

26

cymoxanil mancozeb (248.1 μg/mL). FEAP prevented Botrytis cinerea from infecting

27

excised leaves, intact leaves and isolated fruits of cherry tomato. Its mechanism

28

involved induction of an increase in cell membrane permeability and a decrease in

29

mitochondrial membrane potential.

30 31

KEYWORDS: antifungal protein, purification, plant disease control, fruit preservation,

32

mode

of

2


action

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INTRODUCTION

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Botrytis cinerea is a necrotrophic fungal pathogen that occurs in any place where

35

host plants grow, whether it be a tropical, subtropical or cold temperate region. It infects

36

at least 586 genera of plants including many common crops such as cucumber,

37

strawberry, tomato, potato and grape,1 causing gray mold or other illnesses. Botrytis

38

cinerea infects crops during the various growth periods of crops, but it usually enters

39

host tissues at the early stages of crop development and lurks for a considerable period

40

of time before the advent of a favorable environment and physiological changes in the

41

host. Therefore, not only lesions of stems and leaves and rots of flowers and fruits are

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produced during the growth of the crop, but also the seemingly healthy fruit and

43

vegetable products are seriously damaged during transportation and storage.2, 3 As the

44

second important fungal pathogen in agriculture, Botrytis cinerea has caused huge

45

economic losses in agricultural production, and it has also incurred an enormous

46

amount of expenditures for its disease management every year.3 At present, the disease

47

control of Botrytis cinerea mainly depends on chemical control,4 but the use of

48

chemical agents is faced with many challenges today. The occurrence of pathogen

49

resistance reduces the control effect of fungicides,5, 6 and the residues of fungicides also

50

brings safety risks to food and the environment.7, 8 In view of chemical protection facing

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a series of issues, other methods of disease control have emerged. In recent years,

52

biological control and the use of biological control agents have been developed for the

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prevention and control of Botrytis cinerea.9-11 The biological control agents with

54

antifungal activity include oils, phytohormones, antifungal proteins, antifungal peptides 3



55

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and so on.11-13

56

Antifungal proteins and peptides, which represent the first line of defense to

57

protect organisms from microbial attack, exist in animals, plants and microorganisms.14,

58

15 They demonstrate certain application potential in agriculture due to their considerable

59

antifungal activity, safety, and less fungicide resistance.16,

60

proteins and peptides have achieved some success in disease control, grain preservation

61

and improvement of plant variety.18-22 In this study, we purified and characterized a

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protein with inhibitory effect toward Botrytis cinerea from buckwheat seeds, and

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conducted an investigation on its control effect and mechanism of action. Our results

64

indicated that the protein was new. It exhibited repressive activities toward spore

65

germination and mycelial growth in Botrytis cinerea, and effectively protected cherry

66

tomato from infection by Botrytis cinerea. Through this study, we hope to lay a

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foundation for broadening the application of buckwheat antifungal protein and provide

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a stronger theoretical basis for the antifungal protein in the control of Botrytis cinerea.

17

Currently, antifungal

69 70

MATERIALS AND METHODS

71

Materials. Buckwheat seeds were purchased from a local seed market. The

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fungicides carbendazim thiram bromothalonil (20% carbendazim, 10% thiram, 10%

73

bromothalonil) and cymoxanil mancozeb (8% cymoxanil, 64% mancozeb) were

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purchased from a local pesticide store. The Affi-gel blue gel used for chromatography

75

was purchased from Bio-Rad Laboratories, and SP-Sepharose, Mono S 5/50 GL

76

column and Superdex peptide 10/300 GL column were purchased from GE Healthcare. 4


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Botrytis cinerea used in the experiments was provided by Key Laboratory of

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Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University.

79 80

Purification of antifungal protein. Buckwheat seeds (190 g) were added to 1 L

81

of 20 mmol/L NH4OAc buffer (pH 4.6), soaked overnight and then homogenized. The

82

homogenate was centrifuged (10000×g, 4 °C, 30 min). The supernatant was retained as

83

the crude protein extract, and then precooled at 0 °C. Ammonium sulfate was added to

84

the precooled extract to achieve a saturation of 50%, and the extract was centrifuged

85

(10000×g, 4 °C, 30 min) after standing for 4 h. The supernatant was retained, and

86

ammonium sulfate was again added to bring the saturation to 90%. After standing for

87

4 hours, the mixture was centrifuged, and the supernatant was discarded to obtain an

88

active protein precipitate. The protein precipitate was redissolved in 20 mmol/L

89

NH4OAc buffer (pH 4.6) and applied to a cation exchange SP-Sepharose column (5 cm

90

× 16 cm). After the unadsorbed proteins had been washed off with 20 mmol/L of

91

NH4OAc buffer (pH 4.6), the adsorbed proteins were eluted successively with NH4OAc

92

buffer containing 0.2 mol/L, 0.5 mol/L, and 1 mol/L NaCl. The chromatographic

93

fraction with antifungal activity was dialyzed against double-distilled water in a dialysis

94

bag with a molecular weight cut-off of 3500 Da, and then freeze-dried into dry powder.

95

The dry powder was redissolved in 20 mmol/L Tris-HCl buffer (pH 7.5) and applied to

96

an affinity column filled with Affi-gel blue gel. After the unadsorbed proteins had been

97

washed off with 20 mmol/L Tris-HCl buffer (pH 7.5), the adsorbed proteins were eluted

98

with 20 mmol/L Tris-HCl buffer (pH 7.5) containing 1 mol/L NaCl. The 5



99

chromatographic fraction with antifungal activity was dialyzed and lyophilized,

100

redissolved in 20 mmol/L of NH4OAc buffer (pH 4.6), and then loaded onto a Mono S

101

5/50 GL column attached to the AKTA purifier system (GE Healthcare) for cation

102

exchange chromatography using fast protein liquid chromatography (FPLC). The

103

adsorbed proteins were eluted with a gradient of 0-0.16 mol/L NaCl and 1 mol/L NaCl

104

in NH4OAc buffer, and the active component was dialyzed and lyophilized. The protein

105

powder was redissolved in 20 mmol/L of NH4OAc buffer (pH 4.6) and subjected to

106

chromatography on a Superdex peptide 10/300 GL column attached to the AKTA

107

purifier system for gel filtration chromatography, and the proteins were eluted with the

108

same buffer.

109

In each chromatographic step, the antifungal activity of each chromatographic

110

fraction was determined by the following filter paper method. Activated Botrytis

111

cinerea was inoculated to a PDA plate and cultured at 28 °C. When the diameter of the

112

colony reached 3-5 cm, sterile filter papers with a diameter of 6 mm were placed 5 mm

113

from the edge of the colony. A 20 μL aliquot of each eluate was added to the filter

114

paper, and buffer (20 μL) was added as a negative control. The plates were further

115

incubated at 28 °C, and the growth of hyphae at the edge of the colony was observed

116

within 1-2 days.

117

antifungal activity was determined by the BCA method using a kit, and then the yield

118

was calculated.

23

The protein concentration of each chromatographic fraction with

119 120

Molecular weight determination of antifungal protein by tricine-sodium 6


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dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the

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antifungal protein was determined by tricine-sodium dodecyl sulfate-polyacrylamide

123

gel electrophoresis (Tricine-SDS-PAGE) 24 employing 4% stacking gel, 10% spacer gel

124

and 16.5% separating gel were used, and Coomassie brilliant blue dye was used for gel

125

staining at the end of electrophoresis.

126 127

Qualitative identification of antifungal protein and determination of N-

128

terminal amino acid sequence. The antifungal protein samples were electrophoresed

129

as described above, and the gel used for transfer had previously been electrophoresed

130

at 5 mA for 2 h before loading of the sample. After electrophoresis the gel was stained

131

with Coomassie brilliant blue, and the antifungal protein band was excised after

132

destaining of the gel to remove excess stain. Another gel was taken for transferring the

133

sample to a PVDF membrane, followed by staining with Ponceau dye, and the residue

134

was then washed off. The above two samples were sent to Shanghai Applied Protein

135

Technology Co., Ltd for identification and also for determination of N-terminal amino

136

acid sequence. Identification of antifungal protein was performed using matrix-assisted

137

laser desorption/ionization with time-of-flight/time-of-flight mass spectrometry

138

(MALDI-TOF/TOF MS) and the information regarding the resulting peptide fragments

139

was searched by using Mascot 2.2 software to collect qualitative information about the

140

antifungal protein. N-terminal amino acid sequence of the antifungal protein was

141

determined using the Edman degradation method, and the sequence obtained was

142

aligned using the Blast method in the nr database of the NCIB database to gather 7



143

information on proteins similar to the antifungal protein.

144 145

Determination of stability of antifungal protein based on changes in activity.

146

The effects of temperature, acid-base, metal ions and organic solvents on the activity

147

of the antifungal protein were assayed by the filter paper method mentioned above after

148

the antifungal protein had been subjected to different treatments. In the thermostability

149

experiment, 100 μL aliquots of the solution of the antifungal protein were separately

150

exposeed to 20-120 °C for 30 min. Thermal treatments at 0-100 °C were performed by

151

using a water bath, whereas treatments at 120 °C were conducted by using a steam pot.

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In the acid-base stability experiment, 10 μL aliquots of the solution of the antifungal

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protein were separately mixed with 90 μL of PBS buffer (pH 1-14), and left at room

154

temperature for 2 h. In the metal ion stability experiment, 50 μL aliquots of the solution

155

of the antifungal protein were separately mixed with 50 μL of a solution containing KCl,

156

CaCl2, MgCl2, MnCl2 or FeCl3 at a concentration of 10-150 mmol/L, and placed at

157

room temperature for 2 h. In the organic solvent stability test, aliquots of the solution

158

of the antifungal protein were separately mixed with 50 μL of methanol, ethanol,

159

isopropanol or chloroform, and allowed to stand at room temperature for 2 h. In the

160

above experiments, the untreated solution (pH 7) of the antifungal protein was used as

161

the positive control for comparison with the treated solution of the antifungal protein.

162

The final concentration of antifungal protein in all treated samples and positive control

163

was 0.5 mg/mL. In the thermostability and acid-base stability experiments, PBS buffer

164

(pH 7) was used as the negative control. In the metal ion stability experiment, solutions 8


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of KCl, CaCl2, MgCl2, MnCl2 and FeCl3 at the concentration of 150 mmol/L were used

166

as the negative control. In the organic solvent stability experiment, solutions of

167

methanol, ethanol, isopropanol and chloroform in water at the concentration of 50%

168

(v/v) were used as the negative control.

169 170

Determination of antifungal activity of antifungal protein. The half maximal

171

inhibitory concentrations (IC50) of the antifungal protein toward spore germination and

172

hyphal growth in Botrytis cinerea were determined.

173

The method for determining IC50 toward spore germination was as follows.

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Botrytis cinerea was inoculated on PDA medium and cultured for 14 days at 28 °C in

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the dark. The fungal suspension was obtained by adding 5 mL of sterile water to the

176

plate, followed by scraping the surface of the colony with an applicator, and the spore

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suspension was obtained by filtering the fungal suspension through three layers of lens

178

paper twice to remove the hyphae. The spore concentration of the suspension was

179

adjusted to 1×107 /mL and mixed with an equal volume of 0.05% glucose solution and

180

then the mixture mixed with an equal volume of a sterile solution of the antifungal

181

protein at the final concentration of 23.2, 46.4, 92.8, 185.6, 371.2 and 742.4 μg/mL,

182

respectively. A 20 μL aliquot of the final mixture was added to the center of the concave

183

slide which was placed in a petri dish containing two layers of sterile wet filter paper,

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and then incubated at 28 °C for 10 h. In the control group, the solution of the antifungal

185

protein was replaced with PBS buffer (pH 7), and there were 3 replicates in each of the

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control group and the treatment group. In each group, at least 100 spores were observed 9



187

under an optical microscope and the number of germinated spores was recorded. The

188

rate of inhibition of germination of Botrytis cinerea spores by the antifungal protein

189

was calculated by the following formula: germination inhibition rate = (spore

190

germination rate of control group - spore germination rate of treatment group) / spore

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germination rate of control group × 100%, and the IC50 value was calculated by

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employing the SPSS software.

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The method for determining the IC50 toward mycelial growth was as follows. 1200

194

μL of PDA medium (0.7% agar) cooled to 40-50 °C was mixed with 300 μL of a sterile

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solution of the antifungal protein at the final concentration of 33.33, 100, 300, and 900

196

μg/mL, and then added to the culture dish (30 mm × 15 mm). After the medium had

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solidified, the hyphal disk of Botrytis cinerea with a diameter of 6 mm, which was

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severed from the edge of the colony by using a sterile puncher, was inoculated at the

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center of the dish, and each plate was cultured at 28 °C until the colony in the negative

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control group had grown to the edge of the dish. In the negative control group, the

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solution of antifungal protein was replaced by PBS buffer (pH 7). In the positive control

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group, the solution of antifungal protein was replaced by the fungicides carbendazim

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thiram bromothalonil and cymoxanil mancozeb. There were 3 replicates in each of the

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control group and the treatment group. After measuring the colony diameter of Botrytis

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cinerea by the cross method, the rate of inhibition of hyphal growth of the antifungal

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protein against Botrytis cinerea was calculated by the following formula: growth

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inhibition rate = (colony area of control group - colony area of treatment group / colony

208

area of control group) × 100%, and the IC50 was calculated by utilizing the SPSS 10


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software.

210 211

Biocontrol effect of antifungal protein. In order to assay the infectivity of

212

Botrytis cinerea on cherry tomato and the effect of antifungal protein on the growth of

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the pathogen in cherry tomato tissue, detached cherry tomato leaves were used to carry

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out the following experiment. The leaves of one-month-old cherry tomato seedlings

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were soaked in 75% ethanol for 5 s and then washed twice with sterile distilled water.

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After the leaves had dried naturally, they were cut into circular disks with a diameter

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of 1.2 cm using a sterile punch. The circular disks were immersed in sterile water and

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a solution of the antifungal protein at a concentration of 4.59 mg/mL for 5 min, and

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then placed in a petri dish containing two layers of filter paper after the liquid on the

220

leaves had dried. A 10 μL aliquot of a Botrytis cinerea spore suspension at a

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concentration of 1×105 /mL was added to the center of the circular disks immersed in

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sterile water and antifungal protein solution, and the petri dish was then incubated at 28

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°C. After 2 days of culture, the circular leaves of the two groups with different

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treatments were immersed in alcoholic lactophenol trypan blue in a 100 °C water bath

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for 10 min,25 then transferred to alcoholic lactophenol in a water bath for 3 min,

226

followed by decolorizing with chloral overnight, and then placed in a 50% glycerol

227

stock solution. The circular leaves were placed on the slides with the back side facing

228

up, and the growth status of the pathogenic fungus in the leaves was observed under a

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light microscope.

230

In order to demonstrate the effect of antifungal protein on the biocontrol of Botrytis 11



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cinerea, integrated leaves and excised cherry tomato fruits were used to conduct the

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following experiment. Spore suspensions of Botrytis cinerea at a final concentration of

233

1×107 spores/mL containing 2.30 or 4.59 mg/mL of the antifungal protein were

234

prepared separately. Two groups of leaves of one-month-old cherry tomato seedlings

235

were punctured with a needle, followed by inoculation with the above two spore

236

suspensions in a volume of 10 μL. In the negative control group, the spore suspension

237

containing antifungal protein was replaced with a spore suspension (1×107 spores/mL)

238

without antifungal protein, and in the blank control group, spore suspension was

239

replaced with sterile water. In each group, three samples were set. After the seedlings

240

had continued to grow for 9 days, the leaves of each group were photographed by

241

utilizing a camera at the same object distance, and the lesion area of leaves was

242

calculated by the Photoshop software.26 Cherry tomato fruits were soaked in 75%

243

alcohol for 5 s and then rinsed twice with sterile distilled water, followed by puncturing

244

with a sterile needle. Two groups of above fruits were respectively inoculated with the

245

two aforementioned spore suspensions containing antifungal protein in a volume of 10

246

μL. Spore suspension (1×107 spores/mL) without antifungal protein and sterile water

247

were used instead of antifungal protein to treat with the same method as negative

248

control and blank control. In each group, six samples were set. After the inoculated

249

fruits had been placed at 28 °C for 8 days, the wound diameter of each group of fruits

250

was measured. For the experimental data, the significant analysis was performed with

251

the Student t-test.

252 12


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Study of mechanism of action of antifungal protein. The changes of cell

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membrane permeability and mitochondrial membrane potential were observed after

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staining Botrytis cinerea hyphae with the fluorescent dyes SYTOX green and

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Rhodamine 123.27, 28 SYTOX green is a nucleic acid dye that can enter cells when the

257

plasma membrane is damaged. Rhodamine 123 is a cell membrane-penetrating

258

fluorescent dye that can enter the mitochondrial matrix in normal cells and is used as a

259

probe of the mitochondrial transmembrane potential. When it enters the mitochondrial

260

matrix, its fluorescence intensity dwindles or fades away. The well-developed

261

mycelium of Botrytis cinerea cultured on a PDA plate was inoculated into PDA liquid

262

medium or PDA liquid medium containing the antifungal protein at the concentration

263

of 0.2 mg/mL, followed by culture at 28 °C with shaking (150 rpm/min). After 1 day

264

of culture, SYTOX green at a final concentration of 5 μmol/L or Rhodamine 123 at a

265

final concentration of 4 μg/mL were separately added to the fungal suspension, and

266

then allowed to stand in the dark for 30 min. The hyphae were collected and washed

267

with PBS buffer to prepare a smear. The staining of hyphae was observed under a laser

268

scanning confocal microscope.The excitation and emission wavelengths used for

269

observation of SYTOX green staining were 488 nm and 525 nm, and the excitation and

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emission wavelengths used for observation of Rhodamine 123 staining were 561 nm

271

and 595 nm.

272 273 274

RESULTS AND DISCUSSION A highly purified antifungal protein was obtained from the crude protein extract 13



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of buckwheat seeds by employing a protocol that entailed ammonium sulfate

276

precipitation, cation exchange chromatography, affinity chromatography, cation

277

exchange

278

chromatographic fraction SP2 was obtained by cation exchange chromatography of the

279

dissolved precipitate (derived from the crude protein extract by using 50%-90%

280

ammonium sulfate) on an SP-Sepharose column (Fig. 1A). The active chromatographic

281

fraction B3 was subsequently derived from SP2 by affinity chromatography on an Affi-

282

gel blue gel column (Fig. 1B). Cation exchange chromatography of B3 on Mono S

283

yielded the active chromatographic fraction M4 (Fig. 1C). Finally, the active

284

chromatographic fraction SU2 was obtained from M4 by gel filtration chromatography

285

on a Superdex peptide column (Fig. 1D). The yield of the active fraction at each

286

chromatographic step is listed in Table 1. The chromatographic fraction SU2 exhibited

287

a single protein band with a molecular weight of 11 kDa in Tricine-SDS-PAGE (Fig.

288

2), which was named FEAP. FEAP showed a reliable matching score with the

289

buckwheat peptides Fα-AMP1 and Fα-AMP2 by MALDI-TOF/TOF MS analysis, and

290

it also manifested the highest similarity to Fα-AMP1 and Fα-AMP2 by N-terminal

291

sequence alignment. The N-terminal sequence of FEAP was AQXGAQGGGAT (X

292

corresponds to an unknown amino acid), and its similarity with both of the above two

293

buckwheat peptides was 90.91%. The results of mass spectrometric identification and

294

amino acid sequence alignment of FEAP are shown in Tables 2 and 3, respectively.

chromatography

and

gel

filtration

chromatography.

The

active

295

The stability study disclosed that FEAP displayed pronounced thermostability and

296

stability in ambient acidic pH. Its antifungal activity did not undergo a significant 14


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297

decline after exposure to 20-100 °C for 30 min and following incubation in acidic

298

solutions of pH 1-5 for 2 h, but its activity completely vanished after heat treatment at

299

120 °C and after treatment with alkaline solutions of pH 10-14. FEAP also showed

300

favorable stability when confronted with organic solvents: its antifungal activity was

301

retained in the presence of methanol, ethanol, isopropanol or chloroform at the

302

concentration of 50%. The activity of FEAP showed a certain difference in the presence

303

of different metal ions. Its antifungal activity remained untarnished in the presence of

304

K+, Mn2+ and Fe3+ ions at a concentration of 10-150 mmol/L and Ca2+ and Mg2+ ions

305

at a concentration of 10 mmol/L and 50 mmol/L respectively, but its antifungal activity

306

was attenuated in the presence of Ca2+ and Mg2+ ions at 100 mmol/L and higher

307

concentrations. When the concentration of FEAP was 185.6, 371.2 and 742.4 μg/mL,

308

spore germination in Botrytis cinerea was inhibited by 65%, 75% and 91%, respectively

309

(Fig. 3A & Fig. 4A). When the concentration of FEAP was 300 and 900 μg/mL,

310

mycelial growth in Botrytis cinerea was inhibited by 54% and 91% respectively (Fig.

311

3B & Fig. 4B). The IC50 values of FEAP in spore germination and mycelial growth in

312

Botrytis cinerea as calculated by the SPSS software were 79.9 μg/mL and 236.7 μg/mL,

313

respectively. For comparison, the IC50 values of fungicides including carbendazim

314

thiram bromothalonil and cymoxanil mancozeb on the mycelial growth of Botrytis

315

cinerea were 2.4 μg/mL and 248.1 μg/mL, respectively.

316

The excised cherry tomato leaves infected with Botrytis cinerea were observed

317

under the microscope. It was found that Botrytis cinerea spores on leaves soaked in

318

sterile water underwent normal germination, invaded the leaves through the stomata 15



319

(Fig. 5A), and grew extensively inside the leaf tissue (Fig. 5B). On the contrary, Botrytis

320

cinerea spores on leaves soaked in a solution of the antifungal protein failed to

321

germinate (Fig. 5C), indicating that FEAP effectively forestalled Botrytis cinerea

322

infection of excised cherry tomato leaves. When intact cherry tomato leaves were

323

infected with Botrytis cinerea, leaves merely inoculated with Botrytis cinerea spores

324

were severely affected, with lesions spread throughout the leaves (the mean area of

325

lesion was defined as 100%). Leaves inoculated with spore suspension containing 2.30

326

mg/mL of FEAP were also affected, and the mean area of lesion was reduced to 64%

327

(Fig. 6A). However, when the concentration of FEAP in the spore suspension was

328

raised to 4.59 mg/mL, the average lesions area in the inoculated leaves was further

329

reduced to 2.3%. Some of the leaves were not affected and had a similar appearance to

330

the blank control group (Fig. 7A). Botrytis cinerea infection caused serious epidermal

331

invagination and tissue rot in the isolated cherry tomato fruit. In the negative control,

332

the average diameter of the wound in the fruit was 9.1 mm. When the fruits were

333

inoculated with a spore suspension containing FEAP, the severity of the disease in the

334

fruits decreased as the FEAP concentration increased (Fig. 6B). At the FEAP

335

concentration of 2.30 mg/mL, the average diameter of the wound in the fruits was

336

reduced to 4.8 mm. At the FEAP concentration of 4.59 mg/mL, the average diameter

337

of the wound was further reduced to 2.9 mm, and the fruit epidermis at the inoculation

338

site in some fruits was basically smooth (Fig.7B). The aforementioned results revealed

339

that FEAP can effectively thwart Botrytis cinerea infection on integrated leaves and

340

excised cherry tomato fruits. 16


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Fluorescence staining experiments revealed that after staining with SYTOX green

342

and Rhodamine 123, fluorescence appeared in the hyphae of the FEAP-treated group,

343

while fluorescence was indiscernible in the hyphae of the control group (Fig. 8 & Fig.

344

9), indicating that FEAP elicited an increase in cell membrane permeability together

345

with a decline in mitochondrial membrane potential.

346

In this study, we purified an antifungal protein FEAP from the crude extract of

347

Fagopyrum esculentum seeds by using a protocol that comprised ammonium sulfate

348

precipitation and simple chromatographic procedure. The methodology is routine and

349

facile. We obtained 1.1 mg of antifungal protein from 190 g of seeds. It has been

350

reported that a protein and several peptides with antifungal activity have been isolated

351

from Fagopyrum spp., such as protein FtTI, peptides Fα-AMP1 and Fα-AMP2 and

352

another peptide.29-31 Compared with them, the yield of FEAP (0.58 mg per 100 g seeds)

353

was similar to that of Fα-AMP1 (0.6 mg per 100 g seeds), but slightly higher than that

354

of Fα-AMP2 (0.47 mg per 100 g seeds). In terms of molecular mass or protein type,

355

FEAP also has some distinctive characteristics compared with Fα-AMP1 and Fα-AMP2.

356

The molecular weight of FEAP was 11 kDa, while those of Fα-AMP1, Fα-AMP2 and

357

another antifungal peptide were 3879 Da, 3906 Da and 3.9 kDa,29, 30 respectively. FtTI

358

exhibited a molecular mass of 14 kDa when analyzed by SDS-PAGE, and showed two

359

isoforms (11487 and 13838 Da) as analyzed by MALDI-TOF,31 which was closer to

360

that of FEAP. However, the results of mass spectrometric identification and N-terminal

361

sequence alignment show that FEAP is structurally similar to Fα-AMP1 and Fα-AMP2,

362

which belong to the defensin family according to Fujimura et al.29 while FtTI is a 17



363

trypsin inhibitor.

364

Some peptide fragments derived from FEAP match the defensin peptide, thus, we

365

compared FEAP with defensin-like peptides and antifungal proteins from plants. The

366

antifungal activity of FEAP is stable under acidic conditions. Although it has less

367

remarkable pH stability than several defensin-like peptides isolated from buckwheat

368

and other plants, 30, 32, 33 it has certain advantages in thermal stability. It remains stable

369

following heat treatment at 20-100 °C, which is consistent with the findings on the

370

defensin-like peptide NRBAP.33 It is superior in thermostability to defensin-like

371

antifungal peptides from buckwheat (0-70 °C) and brown kidney beans (20-80 °C), and

372

antifungal proteins from cabbage (0-65 °C) and banana (20-50 °C).30, 32, 34, 35 Generally,

373

defensins are rich in disulfides, and convergently utilize double-stranded or triple-

374

stranded beta-sheets crosslinking a disulphide network into a tight core. Disulfide

375

bonding endow defensins with high stability to temperature,36 and the excellent

376

thermostability of FEAP may be associated with this. In addition to its remarkable

377

thermostability, FEAP also has tolerance to organic solvents. FEAP brings about a rise

378

in membrane permeability and a fall in mitochondrial membrane potential. The former

379

is commonly observed in several plant defensin-like peptides and other plant antifungal

380

proteins,33-35, 37, 38 and the latter is similar to a mutant Allium sativum leaf agglutinin

381

(mASAL).27

382

FEAP is isolated from Fagopyrum esculentum. Compared with the reports of

383

Fujimura et al. and Leung and Ng,29,

30

384

progress in research on the antifungal activity of FEAP, and addresses more regarding

the present investigation has made further

18


Page 18 of 34

Page 19 of 34


385

its possible practical application. FEAP not only evinces pronounced stability and

386

demonstrates multiple modes of action, its antifungal activity is also characterized by

387

certain advantageous features. Its IC50 value toward hyphal growth in Botrytis cinerea

388

is lower than the fungicide cymoxanil mancozeb. It is efficacious in preventing Botrytis

389

cinerea infection on cherry tomato. The data signify that FEAP has a promising

390

biocontrol potential.

391

Although FEAP exhibits certain potential for application, research on FEAP has

392

yet to be expanded. Mechanistically, FEAP may suppress fungal growth by

393

upregulating cell membrane permeability and disrupting mitochondrial membrane

394

potential. Cell membrane permeabilization may involve binding to the cell wall,39 and

395

the decrease in mitochondrial potential may further involve apoptosis of fungal cells.40

396

Some proteins have been reported to cause apoptosis in fungi, such as mASAL protein,

397

which leads to loss of mitochondrial membrane potential and accumulation of

398

intracellular reactive oxygen species in Rhizoctonia solani, and induces programmed

399

cell death of mycelial cells.27 The TUBP1 protein isolated from Bacillus axarquiensi

400

brings about mitochondria-mediated apoptotic cell death in Verticillium dahliae,

401

involving a reduction in enzyme activity (mitochondrial dehydrogenases, F0F1-ATPase,

402

malate dehydrogenase, and succinate dehydrogenase), an increase in reactive oxygen

403

species, a decrease in mitochondrial membrane potential, release of cytochrome c and

404

activation of metacaspase.41 Whether FEAP also has the above mentioned effects

405

remains to be verified. In terms of application, it has been reported that the combination

406

of antifungal peptide and chemical fungicide greatly improves the antifungal effect,42 19



407

and the expression of antifungal protein in plants increases the resistance to pathogenic

408

fungi.43 In addition, favorable progress has been made in the expression and production

409

of recombinant antifungal proteins.44, 45 All of the studies mentioned above are feasible

410

directions for further research on the applications of FEAP.

411 412

SUPPORTING INFORMATION AVAILABLE

413

Supplementary figure entitled "A Tricine-SDS-PAGE gel showing purification of

414

FEAP" was provided in Supporting Information. The Supporting Information is

415

available free of charge on the ACS Publications website.

416 417 418 419

FUNDING SOURCES This study was supported by the University-Industry Cooperation Project of Fujian Provincial Department of Science and Technology (2018N5005).

420 421

REFERENCES

422

(1) Elad , Y.; Pertot , I.; Cotes-Prado , A.; Stewart, A., Plant hosts of Botrytis spp.. in:

423

Fillinger S, Elad Y (eds) Botrytis – the fungus, the pathogen and its management in

424

agricultural systems. Springer, Cham 2016.

425

(2) Williamson, B.; Tudzynsk, B.; Tudzynski, P.; van Kan, J. A. L., Botrytis cinerea:

426

the cause of grey mould disease. Mol Plant Pathol 2007, 8 (5), 561-580.

427

(3) Dean, R.; Van Kan, J. A. L.; Pretorius, Z. A.; Hammond-Kosack, K. E.; Di Pietro,

428

A.; Spanu, P. D.; Rudd, J. J.; Dickman, M.; Kahmann, R.; Ellis, J.; Foster, G. D., The 20


Page 20 of 34

Page 21 of 34


429

Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 2012, 13 (4),

430

414-430.

431

(4) Fillinger, S.; Walker, A., Chemical control and resistance management of Botrytis

432

diseases. in: Fillinger S., Elad Y. (eds) Botrytis – the fungus, the pathogen and its

433

management in agricultural systems. Springer, Cham 2016.

434

(5) Saito, S.; Xiao, C. L., Fungicide resistance in Botrytis cinerea populations in

435

California and its influence on control of gray mold on stored mandarin fruit. Plant

436

Disease 2018, 102 (12), 2545-2549.

437

(6) Yin, W. X.; Adnan, M.; Shang, Y.; Lin, Y.; Luo, C. X., Sensitivity of Botrytis

438

cinerea from nectarine/cherry in China to six fungicides and characterization of

439

resistant isolates. Plant Disease 2018, 102 (12), 2578-2585.

440

(7) Esteve-Turrillas, F. A.; Agullo, C.; Abad-Somovilla, A.; Mercader, J. V.; Abad-

441

Fuentes, A., Fungicide multiresidue monitoring in international wines by

442

immunoassays. Food Chem 2016, 196, 1279-86.

443

(8) Herrero-Hernandez, E.; Pose-Juan, E.; Sanchez-Martin, M. J.; Andrades, M. S.;

444

Rodriguez-Cruz, M. S., Intra-annual trends of fungicide residues in waters from

445

vineyard areas in La Rioja region of northern Spain. Environ Sci Pollut R 2016, 23 (22),

446

22924-22936.

447

(9) Vos, C. M. F.; De Cremer, K.; Cammue, B. P. A.; De Coninck, B., The toolbox of

448

Trichoderma spp. in the biocontrol of Botrytis cinerea disease. Mol Plant Pathol 2015,

449

16 (4), 400-412.

450

(10) Haidar, R.; Fermaud, M.; Calvo-Garrido, C.; Roudet, J.; Deschamps, A., Modes 21



451

of action for biological control of Botrytis cinerea by antagonistic bacteria.

452

Phytopathologia Mediterranea 2016, 55 (3), 301-322.

453

(11) Jacometti, M. A.; Wratten, S. D.; Walter, M., Review: Alternatives to synthetic

454

fungicides for Botrytis cinerea management in vineyards. Aust J Grape Wine R 2010,

455

16 (1), 154-172.

456

(12) Yan, J.; Yuan, S. S.; Jiang, L. L.; Ye, X. J.; Ng, T. B.; Wu, Z. J., Plant antifungal

457

proteins and their applications in agriculture. Appl Microbiol Biot 2015, 99 (12), 4961-

458

4981.

459

(13) Leiter, E.; Gall, T.; Csernoch, L.; Pocsi, I., Biofungicide utilizations of antifungal

460

proteins of filamentous ascomycetes: current and foreseeable future developments.

461

Biocontrol 2017, 62 (2), 125-138.

462

(14) Hegedues, N.; Marx, F., Antifungal proteins: more than antimicrobials? Fungal

463

Biol Rev 2013, 26 (4), 132-145.

464

(15) van der Weerden, N. L.; Bleackley, M. R.; Anderson, M. A., Properties and

465

mechanisms of action of naturally occurring antifungal peptides. Cell Mol Life Sci 2013,

466

70 (19), 3545-3570.

467

(16) Szappanos, H.; Szigeti, G. W.; Pal, B.; Rusznak, Z.; Szucs, G.; Rajnavolgyi, E.;

468

Balla, J.; Balla, G.; Nagy, E.; Leiter, T.; Pocsi, I.; Marx, F.; Csernoch, L., The

469

Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on

470

mammalian cells in the intended therapeutic concentration. N-S Arch Pharmacol 2005,

471

371 (2), 122-132.

472

(17) Palicz, Z.; Jenes, A.; Gall, T.; Miszti-Blasius, K.; Kollar, S.; Kovacs, I.; Emri, M.; 22


Page 22 of 34

Page 23 of 34


473

Marian, T.; Leiter, E.; Pocsi, I.; Csosz, E.; Kallo, G.; Hegedus, C.; Virag, L.; Csernoch,

474

L.; Szentesi, P., In vivo application of a small molecular weight antifungal protein of

475

Penicillium chrysogenum (PAF). Toxicol Appl Pharm 2013, 269 (1), 8-16.

476

(18) Gupta, R.; Srivastava, S., Antifungal effect of antimicrobial peptides (AMPs LR14)

477

derived from Lactobacillus plantarum strain LR/14 and their applications in prevention

478

of grain spoilage. Food Microbiol 2014, 42, 1-7.

479

(19) Puig, M.; Moragrega, C.; Ritz, L.; Montesinos, E.; Llorente, I., Controlling brown

480

spot of pear by a synthetic antimicrobial peptide under field conditions. Plant Disease

481

2015, 99 (12), 1816-1822.

482

(20) Lee, I. H.; Jung, Y. J.; Cho, Y. G.; Nou, I. S.; Huq, M. A.; Nogoy, F. M.; Kang, K.

483

K., SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic

484

rice. Plos One 2017, 12 (3).

485

(21) Moosa, A.; Farzand, A.; Sahi, S. T.; Khan, S. A., Transgenic expression of

486

antifungal pathogenesis-related proteins against phytopathogenic fungi-15 years of

487

success. Israel Journal of Plant Sciences 2018, 65 (1-2), 38-54.

488

(22) Wang, W.; Deng, L.; Yao, S.; Zeng, K., Control of green and blue mold and sour

489

rot in citrus fruits by the cationic antimicrobial peptide PAF56. Postharvest Biol Tec

490

2018, 136, 132-138.

491

(23) Yin, C.; Wong, J. H.; Ng, T. B., Isolation of a hemagglutinin with potent

492

antiproliferative activity and a large antifungal defensin from Phaseolus vulgaris cv.

493

hokkaido large pinto beans. J Agr Food Chem 2015, 63 (22), 5439-5448.

494

(24) Schagger, H., Tricine-SDS-PAGE. Nat Protoc 2006, 1 (1), 16-22. 23



495

(25) Frye, C. A.; Innes, R. W., An Arabidopsis mutant with enhanced resistance to

496

powdery mildew. Plant Cell 1998, 10 (6), 947-56.

497

(26) Li, Q. S.; Chen, J. J.; McConnell, D. B.; Henny, R. J., A simple and effective

498

method for quantifying leaf variegation. Horttechnology 2007, 17 (3), 285-288.

499

(27) Ghosh, P.; Roy, A.; Hess, D.; Ghosh, A.; Das, S., Deciphering the mode of action

500

of a mutant Allium sativum leaf agglutinin (mASAL), a potent antifungal protein on

501

Rhizoctonia solani. Bmc Microbiol 2015, 15.

502

(28) Wong, J. H.; Lau, K. M.; Wu, Y. O.; Cheng, L.; Wong, C. W.; Yew, D. T. W.;

503

Leung, P. C.; Fung, K. P.; Hui, M.; Ng, T. B.; Lau, C. B. S., Antifungal mode of action

504

of macrocarpal C extracted from Eucalyptus globulus Labill (Lan An) towards the

505

dermatophyte Trichophyton mentagrophytes. Chin Med-Uk 2015, 10.

506

(29) Fujimura, M.; Minami, Y.; Watanabe, K.; Tadera, K., Purification,

507

characterization, and sequencing of a novel type of antimicrobial peptides, Fa-AMP1

508

and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench.). Biosci

509

Biotech Bioch 2003, 67 (8), 1636-1642.

510

(30) Leung, E. H. W.; Ng, T. B., A relatively stable antifungal peptide from buckwheat

511

seeds with antiproliferative activity toward cancer cells. J Pept Sci 2007, 13 (11), 762-

512

767.

513

(31) Ruan, J. J.; Chen, H.; Shao, J. R.; Wu, Q.; Han, X. Y., An antifungal peptide from

514

Fagopyrum tataricum seeds. Peptides 2011, 32 (6), 1151-1158.

515

(32) Chan, Y. S.; Wong, J. H.; Fang, E. F.; Pan, W. L.; Ng, T. B., An antifungal peptide

516

from Phaseolus vulgaris cv. brown kidney bean. Acta Bioch Bioph Sin 2012, 44 (4), 24


Page 24 of 34

Page 25 of 34


517

307-315.

518

(33) Chan, Y. S.; Ng, T. B., Northeast red beans produce a thermostable and pH-stable

519

defensin-like peptide with potent antifungal activity. Cell Biochem Biophys 2013, 66

520

(3), 637-648.

521

(34) Ye, X. J.; Ng, T. B.; Wu, Z. J.; Xie, L. H.; Fang, E. F.; Wong, J. H.; Pan, W. L.;

522

Wing, S. S. C.; Zhang, Y. B., Protein from red cabbage (Brassica oleracea) seeds with

523

antifungal, antibacterial, and anticancer activities. J Agr Food Chem 2011, 59 (18),

524

10232-10238.

525

(35) Jiao, W.; Li, X.; Zhao, H.; Cao, J.; Jiang, W., Antifungal Activity of an abundant

526

thaumatin-like protein from banana against Penicillium expansum, and its possible

527

mechanisms of action. Molecules (Basel, Switzerland) 2018, 23 (6).

528

(36) Shafee, T. M. A.; Lay, F. T.; Phan, T. K.; Anderson, M. A.; Hulett, M. D.,

529

Convergent evolution of defensin sequence, structure and function. Cell Mol Life Sci

530

2017, 74 (4), 663-682.

531

(37) Lam, S. K.; Ng, T. B., Purification and characterization of an antifungal peptide

532

with potent antifungal activity but devoid of antiproliferative and HIV reverse

533

transcriptase activities from legumi secchi beans. Appl Biochem Biotech 2013, 169 (7),

534

2165-2174.

535

(38) Fang, E. F.; Hassanien, A. A. E.; Wong, J. H.; Bah, C. S. F.; Soliman, S. S.; Ng,

536

T. B., Purification and modes of antifungal action by Vicia faba cv. egypt trypsin

537

inhibitor. J Agr Food Chem 2010, 58 (19), 10729-10735.

538

(39) van der Weerden, N. L.; Lay, F. T.; Anderson, M. A., The plant defensin, NaD1, 25



539

enters the cytoplasm of Fusarium oxysporum hyphae. J Biol Chem 2008, 283 (21),

540

14445-14452.

541

(40) Krysko, D. V.; Roels, F.; Leybaert, L.; D'Herde, K., Mitochondrial transmembrane

542

potential changes support the concept of mitochondrial heterogeneity during apoptosis.

543

The journal of histochemistry and cytochemistry : official journal of the Histochemistry

544

Society 2001, 49 (10), 1277-84.

545

(41) Zeng, H.; Li, T.; Tian, J.; Zhang, L. L., TUBP1 protein lead to mitochondria-

546

mediated apoptotic cell death in Verticillium dahliae. Int J Biochem Cell B 2018, 103,

547

35-44.

548

(42) Taveira, G. B.; Mello, E. O.; Carvalho, A. O.; Regente, M.; Pinedo, M.; de La

549

Canal, L.; Rodrigues, R.; Gomes, V. M., Antimicrobial activity and mechanism of

550

action of a thionin-like peptide from Capsicum annuum fruits and combinatorial

551

treatment with fluconazole against Fusarium solani. Biopolymers 2017, 108 (3).

552

(43) Vasavirama, K.; Kirti, P. B., Constitutive expression of a fusion gene comprising

553

Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein

554

(RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco. Plant

555

Cell Tiss Org 2013, 115 (3), 309-319.

556

(44) Chowdhury, S.; Basu, A.; Kundu, S., Cloning, Characterization, and bacterial

557

over-expression of an osmotin-like protein gene from Solanum nigrum L. with

558

antifungal activity against three necrotrophic fungi. Mol Biotechnol 2015, 57 (4), 371-

559

381.

560

(45) Sonderegger, C.; Galgoczy, L.; Garrigues, S.; Fizil, A.; Borics, A.; Manzanares, 26


Page 26 of 34

Page 27 of 34


561

P.; Hegedues, N.; Huber, A.; Marcos, J. F.; Batta, G.; Marx, F., A Penicillium

562

chrysogenum-based expression system for the production of small, cysteine-rich

563

antifungal proteins for structural and functional analyses. Microb Cell Fact 2016, 15.

564 565

FIGURE CAPTIONS

566

Figure 1. Elution profiles of antifungal protein FEAP: A: cation exchange

567

chromatography of proteins precipitated by 50%-90% ammonium sulfate on SP-

568

Sepharose column; B: affinity chromatography of component SP2 on Affi-gel blue gel

569

column; C: cation exchange chromatography of component B3 on Mono S column; D:

570

gel filtration chromatography of component M4 on Superdex Peptide column.

571

Figure 2. Molecular weight determination of FEAP by Tricine-SDS-PAGE.

572

Figure 3. Inhibition of spore germination and mycelial growth in Botrytis cinerea

573

by FEAP: A: inhibition rate of spore germination; B: inhibition rate of mycelial growth.

574

Figure 4. Inhibitory effects of FEAP toward spore germination and mycelial

575

growth in Botrytis cinerea: A: spore germination status; B: mycelial growth status;

576

numerical value indicates the concentration (μg/mL) of FEAP in the medium.

577 578

Figure 5. Growth status of Botrytis cinerea in excised cherry tomato leaves: A, B: leaves were soaked in sterile water; C: leaves were soaked in FEAP solution.

579

Figure 6. Incidence of infection in cherry tomato: A: relative lesion area in intact

580

leaves; B: wound diameter of excised fruit; numerical value indicates the concentration

581

(μg/mL) of FEAP, * indicates the significance at P < 0.05.

582

Figure 7. Effects of FEAP on prevention of Botrytis cinerea infection of cherry 27



583

tomato: A: prevention effect in intact leaves; B: prevention effect in excised fruit;

584

numerical value indicates the concentration (μg/mL) of FEAP.

585 586 587 588

Figure 8. FEAP elicited an increase in cell membrane permeability of Botrytis cinerea observed by SYTOX green staining. Figure 9. FEAP elicited a decrease in mitochondrial membrane potential of Botrytis cinerea observed by Rhodamine 123 staining.

28


Page 28 of 34

Page 29 of 34


TABLES Table 1 Yields of active components in different chromatographic steps Purification stage

Fraction

Yield (mg)

Ammonium sulfate precipitation

50-90% NH4SO4

1184

Cation exchange chromatography

SP2

418

Affinity chromatography

B3

303

Cation exchange chromatography

M4

26.9

Gel filtration chromatography

SU2

1.1

Table 2 MALDI-TOF/TOF MS results of peptide fragments derived from FEAP Protein

Fa-AMP1

Fa-AMP2

Species

Fagopyrum esculentum

Fagopyrum esculentum

YCGAGCQSNCK;

YCGAGCQSNCR;

AQCGAQGGGATCPGG

AQCGAQGGGATCPGG

LCCSQWGWCGSTPK

LCCSQWGWCGSTPK

Protein MW

3879.1 Da

3906.1 Da

Sequence IDa

P0DKH7.1

P0DKH8.1

100

99.5

Matched fragment

Protein score C. I. %b

a: according to the Sequence ID, more information of the peptides can be obtained from the protein database of the NCIB database; b: It represents the matching degree of FEAP with Fa-AMP1 and Fa-AMP1, and the value >95 indicates that the result is reliable. Table 3 Alignment results of the N-terminal amino acid sequence of FEAP Protein Species

FEAP

Fa-AMP1

Fa-AMP2

Fagopyrum

Fagopyrum

Fagopyrum

esculentum

esculentum

esculentum

AQCGAQGGGAT

AQCGAQGGGAT

CPGGLCCSQWG

CPGGLCCSQWG

WCGSTPKYCGA

WCGSTPKYCGA

GCQSNCK

GCQSNCR

3879.1 Da

3906.1 Da

P0DKH7.1

P0DKH8.1

91%

91%

AQXGAQGGGAT Sequence

(N-terminal sequence)

Protein MW Sequence IDa Identitiesb

11 kDa

a: according to the Sequence ID, more information of the peptides can be obtained from the protein database of the NCIB database; b: it indicates the similarity of amino acid sequence of FEAP compared with Fa-AMP1 and Fa-AMP2.

29



Page 30 of 34

FIGURE GRAPHICS Figure 1

0.3

SP2

1.0

0.2 0.5

0.1 0.0 0

800

1600

2400

3200

0.4

1.5

B3 0.3

1.0

0.2 0.5

0.1 0.0

0.0 4000

0.0 0

600

1.5

1.0

1.0 0.5

0.5 0.0

Absorbance at 280 nm

D

1.5

M4 Conc. of NaCl (M)


2.0

5

10

15

2400

3000

0.05

SU2

0.04 0.03 0.02 0.01 0.00

0.0 0

1800

Elution volume (mL)

Elution volume (mL)

C

1200

0

20

5

10

15

Elution volume (mL)

Elution volume (mL)

Figure 2 FEAP Markers kDa 40 25

15

10

4.6 1.7

30


20

25

Conc. of NaCl (M)

B

1.5


0.4

Conc. of NaCl (M)


A

Page 31 of 34


Figure 3

A Inhibition rate (%)

100 75 50 25

74 2. 4

18

37 1. 2

5. 6

.8 92

.4 46

23 .2

0

Conc. of Protein (g/mL)

B Inhibition rate (%)

100 80 60 40 20

90

30

0. 0

0. 0

0

0

0 10 0. 0

33

.3 3

0

Conc. of Protein (g/mL)

Figure 4 A Negative control

B

Negative control

Treatment 23.2

46.4

92.8

185.6

371.2

742.4

Treatment 33.33

100

31


300

900


Figure 5 A

B

C

32


Page 32 of 34

Page 33 of 34


Figure 6

Relative lesion area (%)

200

B

*

Wound diameter (mm)

A

150

* 100 50 0

Control

2.30

15

*

10

5

0

4.59

Control

Treatment

2.30

4.59

Treatment

Figure 7 A

Negative control

B

Negative control

Treatment 4.59

2.30

Treatment 4.59

2.30

33


Blank control

Blank control


Figure 8 Control

Treatment

Control

Merger Bright field Dark field

Treatment

Merger Bright field Dark field

Figure 9

TOC GRAPHIC

34


Page 34 of 34

A Buckwheat Antifungal Protein with Biocontrol Potential to Inhibit

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