Watery Saliva Secreted by the Grain Aphid Sitobion avenae

Subscriber access provided by Caltech Library

Article

Watery saliva secreted by the grain aphid Sitobion avenae stimulates aphid resistance in wheat Yong Zhang, Jia Fan, Frederic Francis, and Julian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03141 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

Watery saliva secreted by the grain aphid Sitobion avenae stimulates aphid resistance in wheat *

*

Yong Zhang1,2, Jia Fan1, Frédéric Francis2 , Julian Chen1

1 State Key Laboratory of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of

Agricultural Sciences, Beijing, 100193, PR China

2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liège, Gembloux, B-5030,

Belgium

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Infestation with Sitobion avenae induces localized defense responses in wheat; in this

3

study, the role of S. avenae watery saliva in resistance induction was examined by

4

infiltrating aphid saliva into wheat leaves. After feeding S. avenae on an artificial diet

5

for 48 h, we first collected watery saliva from them and then separated the salivary

6

proteins using one-dimensional gel electrophoresis. Gene expression studies showed

7

that infiltration of S. avenae watery saliva in wheat leaves induced strong salicylic

8

acid-responsive defense but moderate jasmonic acid-dependent defense. Feeding on

9

wheat leaves infiltrated with aphid saliva, compared with untreated leaves,

10

significantly decreased the number of nymphs produced per day and the intrinsic rate

11

of increase of the population of S. avenae. In a choice test against untreated wheat,

12

saliva-infiltrated wheat had repellent effects on aphids. Additionally, electrical

13

penetration graph results showed that the feeding behavior of S. avenae on

14

saliva-treated wheat was negatively affected compared with untreated wheat. These

15

findings provided direct evidence that salivary components of S.avenae are involved

16

in the induction of wheat resistance against aphids and further demonstrated the

17

important roles of watery saliva in aphid-plant interactions.

18

KEYWORDS: Sitobion avenae, saliva infiltration, defense responses, aphid

19

performance, choice preference, feeding behavior

20

21 22

2


Page 2 of 37

Page 3 of 37


23

INTRODUCTION

24

During the long course of their co-evolution with insects, plants have evolved a

25

range of defense mechanisms induced by herbivore attacks, including direct defenses

26

and indirect defenses. Direct defenses include herbivore-induced toxic secondary

27

metabolites and proteinase inhibitors (PIs) that have negative effects on insect

28

development, while indirect defenses consist of volatile emissions that repel

29

herbivores or attract their natural enemies and are released by plants in response to

30

herbivore feeding 1, 2.

31

Jasmonic acid (JA) and salicylic acid (SA) function as two important signaling

32

molecules in the induction of plant defense responses 3. Current theory posits that

33

plants respond to necrotrophic pathogen infestations and leaf-chewing herbivores by

34

activating the JA-mediated defense pathway and that SA-dependent defenses are

35

mainly triggered by biotrophic pathogens and phloem feeders 4.

36

As one of the largest groups of phloem-feeding insects, aphids (Hemiptera:

37

Aphidoidea) are economically important pests that cause heavy losses in agriculture

38

and horticulture worldwide 5. The induction of a SA-dependent defense pathway by

39

aphid feeding has been demonstrated in many aphid-plant interactions, for example,

40

green peach aphid (Myzus persicae) in tomato, tobacco and Arabidopsis

41

Russian wheat aphid (Diuraphis noxia) in wheat 9. However, several genes involved

42

in the jasmonic acid signaling pathway, such as lipoxygenase (LOX) and PIs, were

43

also found to be induced by the feeding of the potato aphid (Macrosiphum euphorbiae)

44

on tomato in compatible and incompatible interactions 3


10

6-8

and

, the greenbug aphid


45

(Schizaphis graminum) feeding on sorghum

46

Arabidopsis 12.

Page 4 of 37

11

, and M. persicae feeding on

47

Plants have the ability to perceive herbivore-derived chemical cues in saliva,

48

such as herbivore-associated elicitors or herbivore-associated molecular patterns

49

(HAMPs), to activate specific defense responses at a minimal fitness cost

50

salivary elicitors or HAMPs have been identified in chewing insects, including fatty

51

acid-amino acid conjugates, glucose oxidase, inceptins in lepidopterans, and

52

disulfooxy fatty acids in the American grasshopper (Schistocerca americana), all of

53

which can induce the activation of SA-, JA-, ethylene (ET)- and reactive oxygen

54

species (ROS) defense responses in plants 14-18.

13

. Many

55

During the process of probing and feeding, aphids initially secrete gelling saliva

56

that can solidify into a tube-like sheath to protect the stylets from mechanical damage

57

and chemical attacks

58

mixture of enzymes and other defense-eliciting components, into the plant cells and

59

apoplasts 20. It has been proposed that aphid interactions with plant immunity involve

60

a gene-for-gene model in which some eliciting components or elicitors can be

61

recognized by nucleotide binding site-leucine rich repeat (NBS-LRR) resistance (R)

62

protein in plants leading to resistance against aphids

63

specific elicitors have been identified from aphids, the eliciting activity of watery

64

saliva in plant defense has been well demonstrated in M.persicae. Infiltration of M.

65

persicae salivary components in the range of 3-10 kDa into Arabidopsis plants

66

activated resistance against aphids resulting in reduced fecundity, and 52 genes such

19

. Additionally, aphids inject watery saliva, a more complex

8, 21

4


. Although, to date, no

Page 5 of 37


67

as senescence associated protein 1 and cytochrome P450, involved in stress responses

68

were also induced as being activated after aphid feeding

69

oxidative enzymes, such as pectinases and polyphenol oxidase (PPO), were also

70

detected in aphid saliva, and have been shown to trigger plant defense responses as

71

eliciting agents

72

salivary elicitors of M. persicae, reduced aphid fecundity, while Mp10 induced

73

chlorosis and activated the SA and JA signaling pathways in Nicotiana benthamiana,

74

all of which indicates their important roles in plant defense induction 26, 27.

22, 23

. Several hydrolytic and

20, 24, 25

. The overexpression of Mp10 and Mp42, two candidate

75

The grain aphid, Sitobion avenae, is one of the most dominant and destructive

76

pests of wheat in the world in that it both feeds directly on phloem sap and transmits

77

barley yellow dwarf viruses 28. The feeding of S. avenae increased the enzyme activity

78

of LOX, PPO, phenylalanine ammonia lyase (PAL) and β-1,3-glucanase (BGL2)

79

related to both the JA and SA pathways in wheat, as well as the mRNA levels of

80

allene oxide synthase (AOS) and PAL, which are involved in JA and SA synthesis,

81

respectively

82

S.avenae 30, little is known about the roles of saliva in aphid-wheat interactions. In our

83

study, the watery saliva of S. avenae was collected and then infiltrated into wheat

84

leaves to investigate the roles of aphid saliva in the induction of wheat defense and

85

the attendant effects on aphid performance by reverse transcription quantitative

86

real-time PCR (RT-qPCR), bioassays and electrical penetration graph (EPG)

87

recording.

88

MATERIALS AND METHODS

29

. Although some proteins have been identified in the watery saliva of

5



89

Insects and plants

90

Winter wheat seeds, Triticum aestivum var. Beijing 837, were immersed in 0.5 %

91

sodium hypochlorite (Amresco, OH, USA) for 30 min to sterilize the surface, then

92

washed 3 times in distilled water. These seeds were transferred into sterilized petri

93

dishes and germinated in distilled water for 3-4 days at a temperature of 25±1 °C; the

94

water was changed every day. Healthy seedlings of similar size were carefully

95

transferred into plastic pots with organic soil (peat: vermiculite=3:1) and continued to

96

be reared in the climate chamber until the two-leaf stage for use in further study (L:

97

D=16 h: 8 h; 20±1 °C).

98

A clone of S. avenae was initially established from a single aphid collected from

99

a wheat field in Langfang City, Hebei Province, North China, and has been reared on

100

wheat plants (Beijing 837 variety) for 6 yrs (25-30 generations every year) in an

101

indoor environment with a temperature of 20±1 °C, relative humidity of 40-60 % and

102

a photoperiod of L: D = 16 h: 8 h.

103

Aphid infestation treatments

104

At the two-leaf stage, 20 wingless adults of S. avenae were transferred to the first

105

leaf (the older leaf) of wheat and the movement of aphids was restricted in a plastic

106

ecological cage (2.7×2.7×2.7 cm) to avoid the escape of aphids. The edge of the

107

ecological cage was covered with sponge to avoid causing mechanical wounds to the

108

leaf. This aphid feeding site was designated the “local leaf” group. The other leaf of

109

the same plant was also caged without aphids as the “systemic leaf”. In the control

110

plant, both leaves were caged at corresponding sites with ecological cages containing 6


Page 6 of 37

Page 7 of 37


111

no aphids. Each pot contained one wheat plant and was kept in a climate incubator

112

with a temperature of 20 ± 1°C and a photoperiod of 16 h: 8 h (L: D). After 30 min,

113

all aphids had begun settling and feeding; this time was recorded as 0 h. After 48 h of

114

feeding, all aphids were removed, and leaf samples were then collected. Three

115

experimental replicates were conducted for each treatment.

116

Aphid saliva collection and 1D gel electrophoresis Chemically defined diets for S. avenae were formulated as previously described

117

118

31

and sterilized with 0.22 µm Millipore membrane filters (Merck Millipore,

119

Germany). Then, 1 mL of artificial diet was sandwiched between two layers of

120

Parafilm membrane (Bemis, WI, USA) stretched across a PVC tube, 27 mm in

121

diameter and 40 mm high, under sterile conditions. The Parafilm was sterilized and

122

exposed to UV light for a minimum of 1 h before use. Approximately 200 S. avenae

123

of different instars were carefully collected from wheat plants and starved for 2 h, then

124

transferred to the PVC tubes to feed on the artificial diet for 48 h in an environmental

125

chamber (20±1°C, L: D=16 h: 8 h); tubes with the same volume of artificial diet but

126

without aphids feeding were used as a control. The secreted saliva was collected from

127

a total of 100 mL of diet (approximately 20,000 aphids) and stored at -70°C until use.

128

The salivary sample was concentrated to a volume of 2 mL using a Vivaspin 20

129

centrifuge concentrator (Sartorius, Gottingen, Germany) with a 3000 Da molecular

130

weight cut-off PES membrane at 4 °C, 15000 g for at least 1 h. Ten microliters of

131

concentrated saliva sample or 5 µL of protein ladder (PageRulerTM Unstained Protein

132

Ladder, Thermo Scientific, USA) mixed with an equal volume of loading buffer was 7



133

heated in boiling water for 5-10 minutes then loaded into the wells of the gel. Proteins

134

were separated by one-dimensional polyacrylamide gel electrophoresis with 5%

135

stacking and 12 % separating gel. Silver nitrate staining was conducted as previously

136

described to detect aphid salivary protein bands 32.

137

Saliva infiltration

138

Twenty microliters of concentrated saliva or control sample was diluted to a

139

volume of 200 µL with distilled water and then infiltrated into the first leaf of a wheat

140

plant at the two-leaf stage using a 1 mL syringe without the needle. Leaves infiltrated

141

with same volume of control sample were used as control groups. The plants were

142

then reared in the same environment as described above for further study. The

143

infiltrated leaves of the plants were collected after 6 h and 24 h of infiltration.

144

Total RNA isolation and cDNA synthesis

145

Leaf samples were collected using sterilized scissors and transferred into liquid

146

nitrogen immediately, then stored at -70 °C until use. Total RNA was extracted from

147

leaves using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) following the

148

protocols provided by the manufacturer. The quality and quantity of RNA were

149

assessed with NanoDrop™ 2000 Spectrophotometers (Thermo Scientific, CA, USA).

150

A total of 1 µg of RNA was reverse transcribed into cDNA with a Transcript One-Step

151

gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing,

152

China) following the manufacturer’s instructions, and cDNA templates were stored at

153

-20°C until they were used for RT-qPCR.

154

RT-qPCR analysis 8


Page 8 of 37

Page 9 of 37


155

The relative expression of genes involved in the JA and SA defense signaling

156

pathways of wheat after aphid infestation and saliva infiltration were detected using

157

RT-qPCR. Target genes for the JA-responsive pathway included LOX, AOS and Ω-3

158

fatty acid desaturase (FAD) , which are involved in JA biosynthesis

159

tested for the SA-responsive pathway were the SA synthesis enzymes PAL and

160

isochorismate

161

pathogenesis-related protein 1 (PR-1) 34. Actin was used as an internal control and

162

was synthesized according to Liu et al. 33. Primers for RT-qPCR were designed using

163

Primer Premier 5.0. All primer sequences are shown in Table 1.

synthase

(ICS)

and

the

induced

SA

33

. The genes

marker

protein

164

RT-qPCR was performed on an ABI 7500 Real-Time PCR System (Applied

165

Biosystems, Carlsbad, CA, USA). cDNA was diluted 10-fold and then used as

166

templates to detect the relative expression of the target genes in a 20 µL reaction

167

system containing 2 µL of cDNA, 0.5 µL each of 10 µmol L-1 forward primer and

168

reverse primer, 10 µL of 2× SYBR premix Ex TaqTM (Tli RNaseH Plus, Takara,

169

Dalian, China) and 0.4 µL of 50× ROX Reference Dye II (Tli RNaseH Plus, Takara,

170

Dalian, China) under the following conditions: 30 s at 95 °C followed by 40 cycles of

171

30 s at 95 °C and 40 s at 60 °C. In RT-qPCR, there were 3 biological replicates for

172

each treatment, and each replicate consisted of 3 technical replicates.

173

Aphid bioassay

174

Choice test

175

After 24 h of saliva infiltration, wheat plants were placed horizontally on the flat

176

table, and the same length of leaves (5 cm) with two different treatments were 9



Page 10 of 37

177

carefully inserted into a transparent plastic column (24 cm in width, 5 cm in height)

178

from holes in opposite sides (Figure 1). Thirty winged S. avenae were collected in a

179

2.0 mL centrifuge tube and then released from the middle of plastic column device.

180

The number of aphids on each leaf was recorded at 6, 24 and 48 h. There were 15

181

replicates for each test, and all of them were conducted in environmentally controlled

182

room with a temperature of 20±1°C, relative humidity of 40-60 % and a photoperiod

183

of L:D=16 h:8 h.

184

Intrinsic rate of increase of aphid population

185

At the two-leaf stage, the first wheat leaves were infiltrated with aphid saliva as

186

described above. After 24 h, one newborn aphid was transferred onto the

187

saliva-treated wheat leaf and the movement of aphids was restricted on the leaf in a

188

plastic ecological cage (2.7×2.7×2.7 cm). The edge of the ecological cage was

189

covered with sponge to avoid causing mechanical wounds to the leaf. The instar of the

190

aphid was checked every 12 h to record the time when it produced the first nymphs.

191

Then, the number of newborn nymphs was recorded every day, and the nymphs were

192

removed after each count to avoid crowding. The period from the birth of the aphid to

193

its first reproduction was defined as development days (Td). The number of newborn

194

nymphs during the Td was expressed as Md. The intrinsic rate of increase (rm) for

195

each aphid was calculated by the following equation: rm=0.738 × (lnMd) / Td

196

Fifteen replicates were conducted in each group. The wheat seedlings were replaced

197

every 3 days with new seedlings receiving the same treatment.

198

Mean relative growth rate of aphid 10


35

.

Page 11 of 37


199

Fifteen newborn aphid nymphs were collected into 0.2 mL microcentrifuge tubes

200

and weighed, and then all aphids were fed on saliva-treated or control wheat leaves as

201

described above. After 7 days, all 15 aphids were collected and weighed again. The

202

wheat seedlings were replaced every 3 days with new seedlings receiving the same

203

treatment. Each pot contained one wheat plant and was kept in a climate incubator

204

with a temperature of 20±1°C and a photoperiod of 16 h: 8 h (L:D). A total of 18

205

replicates were performed for each treatment. The mean relative growth rate (MRGR)

206

of S. avenae was calculated as described previously: MRGR=(ln 7-day weight - ln

207

birth weight)/7 36.

208

Detection of aphid feeding behavior by EPG

209

EPG (Giga-8d) was conducted to record the feeding behavior of S. avenae on

210

wheat leaves. Wingless adult S. avenae were gently collected from wheat plants using

211

a brush and starved for 30 min, then 2-3 cm of 18 mm gold wire was attached to the

212

abdomen of each aphid with water-based silver glue. The plant electrode was inserted

213

into the soil in the pot in order to obtain successful electrical access. The complete

214

insect electrode was carefully pinned into the probe input (BNC connector). EPG was

215

performed from 10:00 to 16:00 every day and recorded continuously for 6 h. Each

216

aphid and plant was used only once. All experiments were carried out in a Faraday

217

cage at 20±1°C. The visualization and manual labeling of the various feeding waves

218

were carried out using Stylet+d. Characteristics of the aphid feeding waves were

219

identified as described in a previous study 37, 38.

220

Statistical analysis 11



221

All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, USA).

222

The percentages of S. avenae that settled on plant leaves in the choice test were

223

arcsine-square-root transformed before analysis, and the differences between groups

224

were examined using Student’s t-test. EPG data were analyzed by a Mann-Whitney U

225

test. For RT-qPCR, each treatment was performed in triplicate, and the differential

226

expression was calculated using 2–∆∆CT method 39. The fold change of the expression

227

of genes involved in the JA and SA signaling defense pathways between the control

228

and treatment conditions was calculated and then analyzed using Student’s t-test. P

229

values less than 0.05 were considered statistically significant.

230

Results

231

Aphid feeding induced local defense responses

232

To determine whether aphid infestation could induce resistance in wheat, two

233

genes involved in JA- and SA-mediated defense responses were identified as

234

differentially expressed after S. avenae feeding (Figure 2A and 2B). The relative

235

expression of the JA-responsive gene FAD had a significant increase in local leaves

236

after infestation by aphids (1.98±0.092-fold, t4=5.745, P=0.005), but in systemic

237

leaves of aphid-infested plants, the mRNA levels of FAD were not significantly

238

different from those in uninfested plants. Similarly, the relative expression of the

239

SA-responsive gene PR-1 was significantly up-regulated in local leaves that had been

240

fed upon by aphids previously (4.04±0.88-fold, t4=4.865, P=0.008), whereas no

241

significant differences were observed in the systemic leaves of the same plants. These

242

results indicated that aphid feeding induced a local defense response in wheat. 12


Page 12 of 37

Page 13 of 37


243

Collection of S. avenae saliva

244

Aphid salivary protein bands were detected on 12 % separated gel using silver

245

staining. The results in Figure 3 show that the protein bands were obviously stained

246

and were mainly distributed at approximately 15 kDa and between 40 kDa and 85

247

kDa.

248

Expression of defense-related genes after S. avenae saliva infiltration

249

Some key genes involved in the JA and SA defense pathways were found to be

250

differentially expressed in wheat leaves (Figure 4A and 4B). The three JA-responsive

251

genes showed no significant difference between treatment and control conditions after

252

6 h of infiltration, but the SA synthesis enzyme PAL and the SA downstream signaling

253

protein PR-1 were significantly up-regulated, with 2.94±0.76-fold (t2.009=4.441,

254

P=0.047) and 4.17±0.73-fold (t4=4.477, P=0.011) increases, respectively.

255

After 24 h of saliva treatment, the relative expression of the JA defense-related

256

gene AOS increased significantly (1.94±0.42-fold; t4=3.791, P=0.02) compared with

257

the level found in the control, but there was no significant difference in the expression

258

of FAD (t4=1.216, P=0.291). The expression level of LOX was also up-regulated

259

1.57±0.15-fold, showing a significant increase (t4=3.076, P=0.037) between the saliva

260

and control treatments. The mRNA levels of both PAL (5.39±1.59-fold; t4=4.36,

261

P=0.012) and ICS (3.07±0.52-fold; t4=6.251, P=0.003), enzymes involved in the SA

262

synthesis pathway, showed significant up-regulation with saliva treatment, and the

263

expression of the SA signaling marker protein PR-1 was up-regulated 14.17±2.71-fold

264

after saliva treatment and showed significant increases compared with the control 13



265

(t2.016=-4.74, P=0.041).

266

Aphid performance

267

The results in Table 2 show that there was no significant difference in the

268

development time or mean relative growth rate of S. avenae between saliva treatment

269

and control. However, the number of nymphs per day produced by S. avenae was

270

reduced significantly when they were fed on wheat leaves with saliva infiltration.

271

Furthermore, the intrinsic rate of increase of the population of S. avenae was also

272

significantly decreased (t28=2.360, P=0.025) after the insects fed on wheat leaves that

273

were infiltrated with aphid saliva compared to the control groups. In the choice test,

274

the percentage of winged aphids that landed on leaves treated with aphid saliva was

275

significantly less than the percentage landing on control groups (t28=6.545, P

Watery Saliva Secreted by the Grain Aphid Sitobion avenae

Recommend Documents