SlERF2 Is Associated with Methyl Jasmonate-Mediated Defense

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Agricultural and Environmental Chemistry

SlERF2 Is Associated with MeJA-mediated Defense Response against Botrytis cinerea in Tomato Fruit Wenqing Yu, Ruirui Zhao, Jiping Sheng, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03971 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Journal of Agricultural and Food Chemistry

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SlERF2 Is Associated with MeJA-mediated Defense Response against Botrytis

2

cinerea in Tomato Fruit

3 4

Wenqing Yu, † Ruirui Zhao, † Jiping Sheng, ‡ and Lin Shen*,†

5 6

†

7

Beijing 100083, China

8

‡

9

China, Beijing 100872, China

College of Food Science and Nutritional Engineering, China Agricultural University,

School of Agricultural Economics and Rural Development, Renmin University of

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* Corresponding Author

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Lin Shen: Tel: +86-10-62737620; E-mail: [email protected]

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1

ACS Paragon Plus Environment


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Abstract

16

Methyl jasmonate (MeJA) and ethylene play important roles in mediating

17

defense responses against Botrytis cinerea. Ethylene response factors (ERFs) are the

18

final components of ethylene signal transduction, whether SlERF2 participates in

19

disease resistance against Botrytis cinerea is unclear. The objective of this study was

20

to investigate the role of SlERF2 in MeJA-mediated defense response by using both

21

sense- and antisense-SlERF2 tomato fruit. Our results showed that both MeJA

22

treatment

23

Overexpression of SlERF2 enhanced tomato fruit resistance against Botrytis cinerea.

24

MeJA treatment increased ethylene production, promoted the activities of Chitinase,

25

β-1,

26

pathogenesis-related proteins content and total phenolic content. Moreover, the effects

27

of MeJA on disease response were reinforced in sense SlERF2 tomato fruit, while

28

weakened in antisense SlERF2 tomato fruit. These results indicated that SlERF2 was

29

involved in MeJA-mediated disease resistance against Botrytis cinerea in tomato fruit.

30

Key words: methyl jasmonate, SlERF2s, defense response, tomato fruit, Botrytis

31

cinerea

and

pathogen

3-glucanase,

infection

phenylalanine

upregulated

ammonia-lyase

SlERF2

and

32 33 34 35 36 2


expression

peroxidase,

level.

elevated

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37

INTRODUCTION

38

Botrytis cinerea (B. cinerea) is an airborne plant pathogen with a necrotrophic

39

lifestyle attacking over 200 plant species, causing grey mold disease 1. Since this

40

fungus is able to infect at low temperatures, it can result in serious economic losses in

41

both pre- and postharvest stages, which has been considered to be a major cause of

42

postharvest rot of perishable plant products 2. Tomato (Solanum lycopersicum) is one

43

of the most widely consumed vegetables, which is susceptible to postharvest infection

44

by B. cinerea, and it has been used as a model for investigating postharvest disease

45

resistance against B. cinerea in fruit 3.

46

Generally, plant defense responses against pathogen attack are mediated by

47

diverse regulatory processes, in which, the classical defense phytohormones, such as

48

salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are known to play key roles

49

4

50

resistance to biotrophic pathogen, whereas JA and ET signaling pathways play

51

important roles in resistance to necrotrophic pathogen, such as B. cinerea 5. Methyl

52

jasmonate (MeJA), a major derivative of JA, is an important endogenous regulator

53

that plays a critical role in inducing resistance to fungal pathogen 6. Increasing

54

evidence showed that exogenous MeJA application could effectively suppress

55

postharvest grey mold disease in various fruits including strawberry 7-8, peach 9, grape

56

10-11

57

promoting activities of defensive enzymes 8-9, and upregulating expression of a series

58

of defense-related genes 7. Moreover, ethylene biosynthesis was induced by MeJA

. It is commonly accepted that SA signaling pathway is necessary for mediating

, and tomato

12-13

, by inducing accumulation of secondary metabolites

3


11, 14

,


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59

treatment in green mature tomato fruit 13, and ERFs expression could also be triggered

60

by MeJA or JA treatment 15. All of the above investigations strongly demonstrated the

61

importance of MeJA in regulation of postharvest fruit immune responses through

62

modification of different signaling networks.

63

Ethylene response factors (ERFs), which are plant specific transcription factors

64

belonging to the large AP2/ERF multi-gene family, are known to act at the last step of

65

ethylene transduction pathway 16. ERF proteins were important in plant responses to

66

both abiotic and biotic stresses by binding to multiple cis-acting elements found in the

67

promoter regions of ET-regulated genes, including the GCC box and DRE/CRT

68

(dehydration responsive element/C-repeat)

69

genes were not only induced by ethylene, but also responded to JA

70

pathogen infection

71

belonging to ERF subfamily

72

biosynthesis 23, and increased chilling tolerance in tomato plant 24. However, it is still

73

unknown whether SlERF2 participated in disease resistance against B. cinerea.

17-18

. Previous studies showed that ERF 19

, SA

20

and

21

. In tomato, 77 genes were postulated to encode proteins 22

. Overexpression of SlERF2 enhanced ethylene

74

Earlier studies using loss-of-function and gain-of-function mutants in different

75

plant species have demonstrated that ERF subfamily members play critical roles in

76

plant response to biotic stresses 25. For instance, overexpression of AtERF1 enhanced

77

expressions of PDF1.2, b-CHI and Thi2.1, resulting in increased resistance to B.

78

cinerea

79

been reported in plants, such as Arabidopsis thaliana

80

canescens

16

. Up to now, ERFs function in pathogen resistance against B. cinerea has

32

, Bupleurum kaoi

33

, and tomato

34

26-30

, grape

31

, Atriplex

. Moreover, most studies on ERFs

4


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81

involvement in defense against B. cinerea infection were performed in Arabidopsis

82

thaliana, such as AtERF1

83

AtERF96

84

response through modulating JA/ET-mediated signaling pathway, resulting in elevated

85

expression of JA-responsive genes such as AtPDF1.2. In tomato, by using

86

virus-induced gene silencing (VIGS)-based method, four members in tomato ERF

87

family were identified to play important roles in resistance to B. cinerea 34. Although

88

the link between ERFs and JA has been documented in Arabidopsis thaliana, little is

89

known about the involvement of SlERF2 in MeJA-mediated disease resistance against

90

B. cinerea in postharvest tomato fruit.

16

, AtERF5

26

, AtERF6

27

, AtERF9

28

, AtERF14

29

, and

30

, which have been proved to function in Arabidopsis thaliana immune

91

Previous study has found that postharvest treatment with MeJA enhanced disease

92

resistance in tomato fruit, and ethylene biosynthesis played a crucial role in

93

MeJA-mediated disease responses. This study presented the role of ethylene signaling

94

component SlERF2 in MeJA-mediated defense responses in postharvest tomato fruit.

95

The objectives of this study were to (i) investigate whether MeJA treatment could also

96

enhance disease resistance in infected tomato fruit, (ii) study whether SlERF2

97

participated in defense response against B. cinerea in tomato fruit, and (iii) explore

98

the roles of SlERF2 in MeJA-mediated defense responses by using both sense- and

99

antisense-SlERF2 tomato fruit.

100 101 102

MATERIALS AND METHODS Fruit Materials, Fungal Cultures. B. cinerea (ACCC 36028) was purchased 5



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from Agricultural Culture Collection of China (Haidian, Beijing). B. cinerea was

104

incubated on potato dextrose agar medium (PDA) and cultured for 7 d at 28 °C. Spore

105

suspensions of the strain were prepared by brushing the surface of culture dishes with

106

0.05 % Tween-80 solution. The spore suspension was adjusted to 1×105 conidia per

107

mL with a hemocytometer 35.

108

Three type tomato fruits (Solanum lycopersicum cv. Zhongshu NO.4) of wild

109

type (WT), sense- and antisense-SlERF2 were harvest at mature green stage from a

110

greenhouse at Xiaotangshan geothermal special vegetable base, Beijing, China

111

(Figure S4). Fruit were immediately delivered to laboratory and were selected

112

according to uniformity shape, color, size, no physical injuries or infections. Twelve

113

hours after picking, all fruit were surface-disinfected with 2 % (v/v) sodium

114

hypochlorite for 2 min, then washed with tap water twice, and air-dried at 25 °C. All

115

fruit were divided into two groups of 130 fruit each for different treatment, and each

116

group was divided into two further categories.

117

For group one, non-inoculated tomato fruit were put in a sealed plastic box,

118

fumigated with either 0 (control) or 0.1 mM MeJA for 12 h (Figure S1 and S4), and

119

then stored at 25 °C, with a relative humidity (RH) of 85−90 %. Ten tomato fruit from

120

each type were sampled at 0.25, 0.5, 1, 3, 6, 9 d after fumigation, and mesocarp

121

tissues from sampled fruit equatorial region were cut into small pieces, frozen in

122

liquid nitrogen rapidly and stored at −80 °C for measurements of SlERF2 gene

123

expression,

124

pathogenesis-related (PR) proteins content. Twenty-four hours after fumigation

defense

enzymes

activities,

total

6


phenolic

content,

and

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125

(Figure S2), ten fruit from each type were inoculation with spore suspension of B.

126

cinerea for measurement of disease symptoms (discussed below).

127

For group two, tomato fruit were wounded with a sterile nail, which made three

128

uniform holes (4 mm deep and 2mm wide) on the equator of each fruit. Then 10 µL

129

spore suspension of B. cinerea was injected into each wound site, all infected fruit

130

were stored at 25 °C with 90–95 % RH

131

pre-inoculated tomato fruit were put in a sealed plastic box, fumigated with either 0

132

(control) or 0.1 mM MeJA for 12 h, and then stored at 25 °C with 90–95 % RH for

133

disease development. Ten tomato fruit from each type were sampled at 0.25, 0.5, 0.75,

134

1, 3, 5 d after fumigation, and mesocarp tissues from sampled fruit equatorial region

135

were cut into small pieces, frozen in liquid nitrogen rapidly and stored at −80 °C for

136

measurements of SlERF2 gene expression, defense enzymes activities, total phenolic

137

content, and PR1 proteins content. Ten pre-inoculated fruit from each type were

138

chosen separately for measurement of disease symptoms.

35

. Twenty-four hours after inoculation,

139

Measurement of Disease Symptoms. Disease incidence and lesion diameter

140

were recorded on the 4th day after fumigation. Disease incidence was expressed as the

141

percentage of inoculation spots showing grey mold symptoms

142

tomato fruit were used only for measurement of disease symptoms.

35

. The inoculated

143

Measurement of Ethylene Content. Ten tomato fruit were taken at 0, 0.5, 1, 3,

144

6, 9, 12 d (group one), and 0, 0.5, 1, 2, 3, 4, 5 d (group two) after MeJA treatments for

145

measurement of ethylene content.

146

Ethylene was assayed by incubating fruit in a 9 L airtight chamber for 1 h at 7



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147

25 °C. A 1mL of headspace gas sample withdrawn from the container using a

148

gas-tight syringe was injected into a gas chromatograph (GC-14C, Shimadzu, Japan),

149

equipped with a GDX-502 column and a flame ionization detector. The column

150

temperature was 50 °C and the injection temperature was 120 °C. The carrier gas was

151

nitrogen with a rate of 50 mL min−1

152

FW (fresh weight) h−1, and all results were replicated three times.

36

. Ethylene content was expressed as nmol g−1

153

Measurement of CHI, GLU, PAL and POD Activities. The activities of CHI,

154

GLU, PAL and POD in tomato fruit were calculated based on fresh weight, and all

155

results were replicated three times. Frozen pericarp tissue of 2.0 g in powder form was

156

homogenized with 5 mL cold extraction buffer: 0.1 M acetic acid buffer (pH 5.2,

157

containing 8 % (w/v) polyvinyl pyrrolidone, 1 mM EDTA and 5 µM

158

β-mercaptoethanol) for Chitinase (CHI, EC 3.2.1.14) and β-1, 3-glucanase (GLU, EC

159

3.2.1.39), 0.2 M boric acid buffer (pH 8.8, containing 10 % (w/v) polyvinyl

160

pyrrolidone, 1 mM EDTA and 5 µM β-mercaptoethanol) for phenylalanine

161

ammonia-lyase (PAL, EC 4.3.1.5), 0.1 M phosphate-buffered saline (pH 7.0) for

162

peroxidase (POD, EC 1.11.1.7). The homogenate was centrifuged at 10,000 ×g for 10

163

min at 4 °C, and aliquot of the supernatant was passed and used for enzyme activity

164

assay. CHI activity was measured based on its ability to decompose chitin, causing the

165

generation of N-acetyl-D-glucosamine as monitored at 585 nm

166

expressed as U·mg−1 FW, where one unit was defined as the formation of 10−9 mol

167

N-acetyl-D-glucosamine produced per hour. GLU activity was assayed by measuring

168

the amount of increased glucose due to hydrolysis of laminarin at 540 nm 8


37

. CHI activity was

35

. GLU

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169

activity was expressed as U·mg−1 FW, where one unit was defined as formation of

170

10−6 mol glucose per hour. PAL activity was measured by following the generation of

171

trans-cinnamic acid at 270 nm 38. PAL activity was expressed as U·mg−1 FW, where

172

one unit was defined as an increase in absorbance of 1 at 290 nm per hour. POD

173

activity was measured by monitoring the increase in absorbance at 470 nm due to

174

oxidation of guaiacol in the presence of hydrogen peroxide

175

expressed as U·mg−1 FW, where one unit was defined as a 1 increase in absorbance at

176

470 nm per minute.

35

. POD activity was

177

Measurement of PR1 Proteins Content. The content of PR1 proteins was

178

measured using an enzyme-linked immunosorbent assay (ELISA) Kit (E0431P1,

179

Huamei, Beijing, China), with polyclonal anti-PR1 antibody (specifically targeted to

180

14-18kD PR1 protein families).

181

Frozen pericarp tissue of 2.0 g in powder form was homogenized with 5 mL cold

182

extraction buffer. Four hours after extraction at 4 °C, the homogenate was centrifuged

183

at 10,000 ×g for 15 min. The supernatant was evaporated under a vacuum, and

184

residue was dissolved in sample diluent before ELISA. According to the ELISA assay

185

process, microtitration plates were coated for 2 h at 37 °C with 100 µL of sample

186

solution. After discarding, 100 µL of solution A were added into plates and coated for

187

1 h at 37 °C, then washed with washing buffer five times. After that, 90 µL of

188

substrate solution was added into each well and incubated for 0.5 h at 37 °C. The

189

absorbance of OD450 nm was read after applying 50 µL of stop bath. PR1 proteins

190

content was expressed as µg g−1 FW. 9



191

Measurement of Total Phenolic Content. Total phenolic content was assayed

192

using the method described by Pirie and Mullins 39. Frozen pericarp tissue of 2.0 g in

193

powder form was homogenized with 10 mL of cold 1 % HCl-methanol (v/v) at 4 °C.

194

Two hours after extraction, the homogenate was centrifuged at 10,000 ×g for 10 min

195

at 4 °C, and aliquot of the supernatant was passed and used for total phenolic content

196

assay. The absorbance was measured at 270 nm with gallic acid as a standard. Total

197

phenolic content was expressed as µg kg−1 FW.

198

Quantitative Real-Time PCR (qRT-PCR).

Total RNA was extracted from

199

0.15 g frozen pericarp tissue using an EasyPure Plant RNA Kit (Beijing Transgen

200

Biotech Co. Ltd., Beijing, China). Remaining genomic DNA was digested by using

201

RNase-free DNase I (Beijing Transgen Biotech Co. Ltd., Beijing, China), according to

202

the manufacturer instruction, then total RNA was dissolved in 30 µL of RNase-free

203

water, and was quantified with a spectrophotometer (NanoDrop Technologies, Inc.).

204

Reverse transcription was completed to synthesize the first-strand cDNA with 2 µg of

205

total RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis

206

SuperMix Kit (Beijing Transgen Biotech Co. Ltd., Beijing, Chia).

207

All qRT-PCR was run with TransStrat Top Green qPCR SuperMix (Beijing

208

TransGen Biotech Co., Ltd, China). 5 µL of SuperMix, 0.3 µL of both the forward and

209

reverse gene specific primers (Table 1), 1 µL of cDNA, and 3.4 µL of RNase-free

210

water were added to a 10 µL final volume per reaction. The thermal cycles processes

211

for PCR amplification were as follows: initial denaturation at 94 °C for 30 s, followed

212

by 40 cycles at 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. SlUbi3 was used as 10


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213

reference housekeeping gene to normalize transcript level for each sample and the

214

final data was calculated using formula 2-△△Ct.

215

Statistical Analysis. All data were presented as the mean ± standard deviation

216

(SD). Significant differences of the means were analyzed by one-way analysis of

217

variance (ANOVA) and Duncan´s multiple range tests using the statistical software

218

SPSS 20.0 (IBM Corp., Armonk, NY).

219 220

RESULTS

221

Effects of MeJA and B. cinerea+MeJA Treatments on SlERF2 Expression.

222

To evaluate the effect of MeJA on SlERF2 expression, SlERF2 expression level was

223

analysis by qRT-PCR. MeJA significantly enhanced expression level of SlERF2 gene

224

in WT and sense-SlERF2 fruit (Figure 1, P < 0.05). However, no significant

225

differences of SlERF2 relative expression were observed in antisense-SlERF2 fruit

226

among control and MeJA treatment, no matter whether fruit were inoculated or not

227

(Figure 1, P > 0.05). Moreover, a smaller increase of SlERF2 expression was

228

observed in MeJA-treated WT fruit, compared to MeJA-treated sense-SlERF2 fruit, in

229

which stronger increases of SlERF2 expression was observed (Figure 1, P < 0.05). In

230

non-inoculated fruit, SlERF2 expression increased gradually and reached a maximum

231

on the 9th day, and SlERF2 expression level in sense-SlERF2 fruit was increased by

232

58.8%, which was 1.92 times higher than those in WT fruit (Figure 1A, P < 0.05). In

233

inoculated fruit, SlERF2 expression level elevated sharply to 2.3- and 1.8-fold in

234

MeJA-treated sense-SlERF2 and WT fruit after 0.5 d and declined gradually (P < 11



235

0.05), but it was still higher than that in control (Figure 1B, P < 0.05).

236

Effects of MeJA+B. cinerea and B. cinerea+MeJA Treatments on Disease

237

Incidence and Lesion Diameter. MeJA treatment could effectively decreased disease

238

symptoms in tomato fruit, before (group one) or after (group two) inoculation with B.

239

cinerea (Figure 2, P < 0.05). In group one, on the 4th day after inoculation, disease

240

incidence of sense-SlERF2 fruit was the lowest, and MeJA treatment had the best

241

inhibition effect on grey mold. In sense-SlERF2 fruit treated with MeJA, disease

242

incidence was inhibited by 37.1 %, which was higher than both in WT fruit of 26.7 %

243

and in antisense-SlERF2 fruit of 6.2 % (Figure 2B, P < 0.05). Similarly, lesion

244

diameters were also smaller in sense-SlERF2 fruit. The lesion diameter of

245

sense-SlERF2 fruit was inhibited by 32.7 %, which was 1.7 times higher than that in

246

MeJA-treated WT fruit (Figure 2A, P < 0.05). Moreover, MeJA treatment after

247

inoculation could also significantly reduce disease symptoms of fruit. The disease

248

incidence was inhibited by 16.3 %, 14.6 %, and the lesion diameter was inhibited by

249

13.8 %, 12.0 % in MeJA-treated WT and antisense-SlERF2 fruit (Figure 2A and B, P

250

< 0.05). These results suggested that SlERF2 played a positive role in MeJA-mediated

251

defense response against invasion and expansion of B. cinerea.

252

Effects of MeJA and B. cinerea+MeJA Treatments on Ethylene Production.

253

Ethylene production in fruit treated with MeJA was elevated compared to that in

254

control during most of the storage period (Figure 3A, C and E, P < 0.05). One peak of

255

ethylene production was observed on the 9th day of storage, and a 31.2 %, 28.3 % and

256

20.8 % elevation was observed in sense-SlERF2, WT and antisense-SlERF2 fruit with 12


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MeJA treatment alone (Figure 3A, C and E, P < 0.05). Moreover, in inoculated fruit,

258

the elevation of ethylene production in sense-SlERF2 fruit was significantly higher

259

than those in WT and antisense-SlERF2 fruit during storage period. The maximum

260

elevation in MeJA-treated fruit was 155.4 % (in sense-SlERF2 fruit), 145.1 % (in WT

261

fruit) and 117.0 % (in antisense-SlERF2 fruit) higher than that in control fruit (Figure

262

3B, D and F, P < 0.05).

263

Effects of MeJA and B. cinerea+MeJA Treatments on CHI, GLU, PAL and

264

POD Activities. MeJA treatment maintained higher CHI, GLU, PAL and POD

265

activities compared with control during the storage period, no matter whether fruit

266

were inoculated or not (Figure 4, P < 0.05). Moreover, overexpression of SlERF2

267

enhanced the effect of MeJA-induced activities of these four enzymes, while silence

268

of SlERF2 lessened this induction (Figure 4, P < 0.05).

269

In non-inoculated fruit, CHI activity in MeJA-treated fruit was significantly

270

higher than those in control fruit. CHI activities were significantly increased by 87.3 %

271

in MeJA-treated sense-SlERF2 fruit, which was 18.4 % and 584.1 % higher than those

272

in WT and antisense-SlERF2 fruit with MeJA treatment on day 6 (Figure 4A, P
0.05).

282

In inoculated fruit, GLU activity shared a trend similar to the MeJA-treated

283

non-inoculated fruit (Figure 4D, P < 0.05). However, MeJA treatment after B. cinerea

284

infection had less effect on GLU activity than treatment with MeJA alone, and no

285

significant differences were observed among control and MeJA-treated both WT and

286

antisense-SlERF2 fruit (Figure 4D, P > 0.05). Overexpression of SlERF2 further

287

promoted the increase of GLU activity in MeJA-treated fruit, and it was 2.7 times

288

higher than the value in control fruit on days 0.5 (Figure 4D, P < 0.05).

289

PAL activity in all non-inoculated fruit displayed a gradually increase and

290

reached a maximum on the 9th day (Figure 4E, P < 0.05). Meanwhile, a 48.4 %, 26.5 %

291

and 7.6 % elevation was observed in sense-SlERF2, WT and antisense-SlERF2 fruit

292

after MeJA treatment (Figure 4E, P < 0.05). Furthermore, in fruit inoculated with B.

293

cinerea followed by MeJA fumigation, PAL activity increased by 109.6 %, 60.4 %

294

and 37.8 % on the 12th hour and 43.6 %, 25.7 % and 9.2 % on the first day of storage

295

in sense-SlERF2, WT and antisense-SlERF2 fruit, compared to fruit inoculated with B.

296

cinerea alone

(Figure 4F, P < 0.05).

297

POD activity was remarkably induced by MeJA treatment both in inoculated and

298

in non-inoculated fruit (Figure 4G and H, P < 0.05). In non-inoculated fruit, POD

299

activity in MeJA-treated fruit peaked on the 6th day then declined, which was 151.9 %

300

(in sense-SlERF2 fruit), 123.6 % (in WT fruit) and 57.1 % (in antisense-SlERF2 fruit) 14


Page 14 of 39

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301

higher than that in control fruit (Figure 4G, P < 0.05). In inoculated fruit, POD

302

activity in MeJA-treated fruit reached a maximum of 25.7 U·g−1 FW (in

303

sense-SlERF2 fruit), 22.9 U·g−1 FW (in WT fruit) and 19.0 U·g−1 FW (in

304

antisense-SlERF2 fruit) on the first day of storage, then declined (Figure 4H, P

SlERF2 Is Associated with Methyl Jasmonate-Mediated Defense

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