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Possible insecticidal mechanisms mediated by immune response related Cry-binding proteins in the midgut juice of Plutella xylostella and Spodoptera exigua Keyu Lu, Yuqing Gu, Xiao-Ping Liu, Yi Lin, and Xiao-Qiang Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05769 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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Journal of Agricultural and Food Chemistry

Possible insecticidal mechanisms mediated by immune response related Cry-binding proteins in the midgut juice of Plutella xylostella and Spodoptera exigua

Keyu Lua, Yuqing Gua, Xiaoping Liua, Yi Lina*, and Xiao-QiangYub* a

Fujian Provincial Key Laboratory of Biochemical Technology (Huaqiao University),

Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao Univiersity, Xiamen 361021, China b

Division of Molecular Biology and Biochemistry, School of Biological Sciences,

University of Missouri - Kansas City, Kansas City, MO 64110, USA *

Corresponding authors: Yi Lin, Ph.D, phone: +86-18965146131, E-mail:

[email protected]; Xiao-QiangYu, Ph.D, [email protected]

1

ACS Paragon Plus Environment


1

Abstract

2

Cry toxins are insecticidal toxin proteins produced by a spore-forming

3

Gram-positive bacterium Bacillus thuringiensis (Bt). Interactions between the Cry

4

toxins and the receptors from midgut BBMVs, such as Cadherin, Alkaline

5

phosphatase (ALP) and Aminopeptidase (APN), are key steps for the specificity and

6

insecticidal activity of Cry proteins. However, little is known about the midgut juice

7

proteins that may interfere with Cry binding to the receptors. In order to validate the

8

hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal

9

process of Cry toxins. We applied Cry1Ab1-coupled Sepharose beads to isolate

10

Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua.

11

Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins

12

in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the

13

Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible

14

insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins:

15

Dorsal and POX-C. Our results suggested that there exist, in the midgut juice,

16

Cry-binding proteins, which are different from brush border membrane vesicles

17

(BBMVs) specific receptors.

18 19

Key words: Bacillus thuringiensis; Cry-binding proteins; Midgut juice; Plutella

20

xylostella; Spodoptera exigua; Immune responses

2


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INTRODUCTION

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Bacillus thuringiensis (Bt) can kill certain insect pests mainly due to the

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parasporal crystal proteins (Cry proteins) they produce. These Bt Cry proteins have

24

been widely used in biopesticide formulations and transgenic crops for insect pest

25

control. 1 There are currently two major modes of actions of Cry toxins: the most

26

accepted model proposed that after ingestion by susceptible insects, Cry toxins are

27

activated by gut proteases and the active toxins bind to specific receptors on midgut

28

epithelial cells.2 Receptor binding induces conformational changes in the toxins,

29

which is necessary for oligomerization of toxin monomers and insertion of toxin

30

oligomers into membrane to form pores, leading to cell lysis and finally to insect

31

death. 2 Another one however proposed that after binding of toxins to receptors, a

32

signal transduction pathway was involved in the insecticidal process.

33

Zhang et al. 4 found that Cry6Aa triggers the Caenorhabditis elegans cell necrosis by

34

an aspartic protease-1 (ASP-1) dependent manner, and Guo et al.

35

Cry1Ac-resistant P. xylostella strains have a constitutively up-regulated MAPK

36

signaling cascade to protect them from cell lysis and death by trans-regulating the

37

expression level of diverse midgut receptor genes, indicating that the mode of actions

38

of Cry toxins are far more complicated than we previously understood.

39

5

3

Recently,

showed that

Cry1Ab proteins are the most studied and applied Cry toxins and insect pests 6

40

have developed resistance to Cry1Ab.

Current available 34 Cry1Ab proteins were

41

classified into four groups based on the predicted 3-dimensional structures of the toxic

42

core, and Cry1Ab1 toxic core shows the identical 3-D structure with that of 28 other

43

Cry1Ab proteins, but three Cry1Ab proteins, Cry1Ab2, Cry1Ab7 and Cry1Ab28,

44

show different 3-D structures from Cry1Ab1.

45

midgut brush border membrane vesicles (BBMVs), such as Cadherin, Aminopeptidase

7

Cry1Ab-binding receptors in the

3



46

N (APN) and Alkaline phosphatase (ALP), have been identified. 8 However, whether

47

there are Cry-binding proteins in insect midgut juice that could affect the toxicity of

48

the toxins remained elusive.

49

To identify the Cry-binding proteins in the insects’ midgut juice and their

50

possible influences on the behaviors of ingested Cry toxins, we performed pull-down

51

assay using a Cry1Ab1-coupled Sepharose beads to identify Cry1Ab1-binding

52

proteins in the midgut juice of two Lepidopteran species: P. xylostella and S. exigua. P.

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xylostella is a Cry1Ab-susceptible insect while Cry1Ab showed low toxicity toward S.

54

exigua (http://www.glfc.forestry.ca/bacillus/). Based on our findings we tempted to

55

explain the possible reasons behind the toxicity difference between the two species

56

and further proposed possible models of actions of Cry toxins.

57 58

MATERIALS AND METHODS

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Bacterial strains and insects. E. coli strain Trans-T1 (TransGen, Beijing, China)

60

and E. coli strain Rosetta (DE3) (TransGen, Beijing, China) were used for gene

61

cloning and expression, respectively. ProteinlsoTM Ni-IDA Resin (TranGen, Beijing,

62

China) was used for purification of recombinant proteins. Eggs of Cry1Ab-susceptible

63

P. xylostella, first instar larvae of S. exigua and their diet were bought from Henan

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Jiyuan Baiyun Industry, Co., Ltd, China.

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Expression of the recombinant Cry1Ab1 protein. The gene of Cry1Ab1 toxic

66

core (GenBank accession number: AAA22330) was cloned into pEASY®-Blunt E1

67

(TransGen, Beijing, China) expression vector to construct a recombinant

68

plasmid-pEASY-Cry1Ab; the recombinant pEASY- Cry1Ab was then transformed

69

into the competent Rosetta (DE3) cells. Positive clones were confirmed by colony

4


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PCR and grown at 37 °C in 10 mL LB medium with 100 µg/mL ampicillin.

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Expression was induced by adding isopropyl b-D-thiogalactoside (IPTG) to a final

72

concentration of 0.1 mM when the OD600 value reached 0.6. Transformants were

73

incubated at 20 °C for 20 h, after which, cells were collected by centrifugation at

74

12,000 rpm, 4 °C for 10 min and disrupted in 20 mM Tris-HCl (pH 8.0) by

75

sonication (3 s on, 5 s off for 30 min). Lysates were clarified by centrifugation at

76

12,000 rpm at 4 °C for 10 min, supernatant were then removed with inclusion bodies

77

remained in pellets. The purification of inclusion proteins were performed according

78

to the method described by Yang et al. .9 Briefly, inclusion bodies were denatured in

79

buffer containing 20mM Tris, 8M Urea, pH 9.5 and were purified by nickel affinity

80

chromatography (ProteinlsoTM Ni-IDA Resin, TransGen, Beijing, China) according to

81

manufactruer’s instructions; flow throughs in each step were collected for SDS-PAGE

82

analysis. Renaturation of proteins were carried out by dialysing denatured inclusions

83

in buffer containing different concentrations of urea (20mM Tris, 2mM GSH, 0.2mM

84

GSSG and 0-6M Urea) successively.

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Bioassay against P. xylostella. The inclusion bodies of Cry1Ab1 were quantified 10

86

using Bradford’s method

and subjected to bioassay against P. xylostella. Cry1Ab1

87

solution (1 mL, μg/g) was mixed with 10 g of artificial diet and was divided into

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three for triplicate use. 1st instar P. xylostella larvae were used in bioassay and were

89

reared on artificial diet treated with Cry1Ab1 in petri dishes(30 larvae per dishes) at

90

25°C with 60% relative humidity, and a 16:8-h photoperiod. Larvae were assayed

91

against different concentrations of Cry1Ab1; Controls groups were treated with the

92

same volume of Tris-HCl (pH.8.0) as that of Cry1Ab1, each treatment was repeated 3

93

times. Mortality was recorded after 48 h. 5



94

Cry1Ab1-coupled SepharoseTM 4B. The coupling process was carried out

95

according to manufacturer’s instructions. Briefly, the purified Cry1Ab1 toxin (1

96

mg/ml) were mixed with CNBr-activated SepharoseTM 4B (GE Healthcare, Fairfield,

97

USA) matrix (2.0 g) in a buffer containing 0.1 M NaHCO3 and 0.5 M NaCl (pH 8.3)

98

on a shaker at 25°C for 6h. Unbound epitopes were blocked by adding 10 mg of

99

glycine. Unbound ligands were washed away with PBS (containing 1M NaCl). The

100

Cry-coupled matrix was stored in 20% ethanol at 4 °C until use.

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Pull-down experiments. To capture Cry1Ab-binding midgut nonreceptor proteins

102

from P. xylostella and S. exigua, pull-down experiments was done with

103

Cry1Ab1-coupled SepharoseTM 4B beads; Sepharose beads without protein ligand

104

coupling were used as control. Midgut lumen juice was collected as follows:

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fourth-instar larvae of P. xylostella and fifth-instar larvae of S. exigua were

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anesthetized on ice for 30 min before midgut tissues were collected. Collected

107

tissues were centrifuged at 7,500 g 4 °C for 10 min, supernatant containing midgut

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lumen juice were subjected to pull-down assay. The pull-down assay was performed

109

according to the method described by Shu et al.,

110

SepharoseTM 4B matrix (100 µl) was incubated with midgut lumen juice (200 µl)

111

for 1 h at 4 °C with each treatment repeated 3 times. Cry-binding proteins were

112

separated by SDS-PAGE and were stained using Fast Silver Stain Kit (Beyotime,

113

Jiangsu, China). After silver staining, pulled down bands were analyzed at Huada

114

Protein Research Center (HPRC) for Liquid chromatography connected tandemly

115

with mass spectrometry (LC-MS/MS) analysis. For P. xylostella, LC-MS/MS data

116

were searched against uni-Plutella_51655 xylostella-51655(785 sequences; 282,669

117

residues) by HPRC using MASCOT search engine (Matrix Science, London, UK).

11

6


briefly, Cry1Ab-coupled

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Search parameters were as follows: type of search: MS/MS Ion Search; enzyme:

119

TrypsinFixed; modifications: Carbamidomethyl (C); variable modifications:

120

Gln->pyro-Glu (N-term Q), oxidation (M); mass values: Monoisotopic; protein mass:

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Unrestricted; peptide mass tolerance: ± 15 ppm; fragment mass tolerance

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20mmu; max missed cleavages: 2; instrument type: Default; number of queries:

123

5,540. For S. exigua, LC-MS/MS data were searched against Lepidoptera_uni_7088

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(265,934 sequences; 69,639,520 residues) by HPRC using MASCOT search engine

125

(Matrix Science, London, UK). Search parameters were as follows: type of search:

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MS/MS Ion Search; enzyme : Trypsin; fixed modifications: Carbamidomethyl (C);

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variable modifications : Gln->pyro-Glu (N-term Q), oxidation (M); mass values:

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Monoisotopic; protein mass : Unrestricted; peptide mass tolerance : ± 15 ppm ;

129

fragment mass tolerance: ± 20 mmu; max missed cleavages: 2; instrument type:

130

Default; number of queries : 11,184.

:

±

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Homology modeling and Molecular docking. The 3-D structure of Cry1Ab1

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(gi:142720), Papilio machaon Peroxiredoxin-4 and S. exigua POX-C were predicted

133

using SWISS-MODEL (http://swissmodel.expasy.org).

134

using the Ramachandran plot by submitting the PDB files to the RAMPAGE server

135

(http://mordred.bioc.cam.ac.uk).

136

(Peroxiredoxin-4 and POX-C) were carried out using Z-dock (Discovery studio 2.5)

137

14

13

12

Models were assessed

Docking between Cry1Ab1 and two proteins

with all parameters set as default.

138 139

RESULTS AND DISCUSSIONS

140

Expression and purification of recombinant Cry 1Ab1 and bioassay. The DNA

141

encoding active Cry1Ab1 was cloned into expression vector pEASY-E1. Positive

142

clones were confirmed by colony PCR. Recombinant plasmids were transformed into 7



143

E. coli Trans-T1 and were then sequenced to make sure the gene sequence was correct.

144

The recombinant pEASY- Cry1Ab1 was transformed into E. coli Rosetta for

145

expression. SDS-PAGE analysis showed that the proteins were expressed inside the

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cells 20h after induction with

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performed bioassay to validate the toxicity of the recombinant protein with 1st instar

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susceptible insect larvae, since they are more sensitive than commonly used 3rd instar

149

larvae. The recombinant Cry1Ab1 was purified (Fig.2) and assayed against 1st-instar

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larvae of P. xylostella. The corrected mortality rates at the concentration of 1 µg/g (1

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g diet containning 1µg recombinant Cry1Ab1) and 128 µg/g were 61.2% and 78.9%,

152

respectively. Mortality rates in the treatment groups were increased after Cry1Ab1

153

treatment compared to control groups (15.5%), indicating that the recombinant

154

Cry1Ab1 is active against P. xylostella.

0.1 mmol/L IPTG, at 20°C （Fig.1）. We then

155

Trypsin-like serine proteases and Dorsal were Cry1Ab1-binding protein in the

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midgut juice of P. xylostella larvae. Cry1Ab1-coupled SepharoseTM 4B beads were

157

used to isolate Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Two

158

bands were observed compared to control (Fig.3). Pulled down bands (Fig.3) were

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analyzed by LC-MS/MS. MASCOT search results with a MASCOT score higher than

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7 (p< 0.05) were taken for further analysis (Table.1). Two trypsin-like serine proteases

161

and Dorsal (Table.1) were identified as putative Cry1Ab1-binding proteins (Fig.3).

162

Another band (Fig.3; Table.1) were identified to contain putative zinc finger protein

163

294 (Danaus plexippus), putative homeodomain transcription factor (Operophtera

164

brumata) and an uncharacterized protein (Bombyx mori), however, no match was

165

found in the P. xylostella genome.

8


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An over-firing immune response might be induced by Cry1Ab1 that leads to Insects’ fat body is an important tissue that is involved in immune

167

insect death.

168

responses against bacterial and fungal pathogens by synthesizing and secreting

169

antimicrobial peptides (AMPs); the production of AMPs is regulated primarily by two

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signaling pathway: the IMD pathway (immune deficiency pathway) and the Toll

171

pathway.

172

The activation of Toll-pathway confers Drosphila on the ability to produce AMPs

173

against fungal and gram-positive bacterial pathogens. 15, 16 Dorsal is one of the three

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NF-κB (nuclear factor- κB; nuclear factor kappa-light-chain-enhancer of activated B

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cells) homologues and is usually trapped in cytoplasm of fat bodies by IκB (inhibitor

176

of κB)- related inhibitor Cactus. 15 When Toll-pathway is activated, Dorsal is released

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due to degradation of Cactus and enters into the nucleus, thus starts the transcription

178

of AMP genes (Fig.4). 15, 16 Pull-down results suggested that the interaction between

179

Cry1Ab1 and Dorsal might affect mutual functions of the two proteins once Cry1Ab1

180

enters the fat body cells. As shown by Cabrera et al. 17 Cry11Aa and Cry1Ab entered

181

the Mos20 cells through both clathrin-dependent and clatherin-independent

182

endocytosis. There are also evidences supporting the transfer of Cry toxins into the

183

cells: sequestration and intergenerational transfer of the Cry toxins were found in two

184

coleopteran insects (Harmonia axyridis

185

Lepidopteran insect (Chlosyne lacinia 20) that fed on preys exposed to Cry toxins. It

186

was speculated that once the Cry protein crossed the peritrophic matrix, it might be

187

stored in the perivisceral fat body, from there it might be transported to oocytes.

188

Notably, a fat body APN was identified to interact with Cry1A toxins in moth Achaea

189

janat. This fat body specific APN showed different interaction patterns with various

190

Cry1A toxins compared to that of the BBMVs specific APN.

15

Dorsal is a key component of the Toll-pathway in Drosphila fat bodies.

18

and Propulae japonica

9


21

19

) and one

20

Thus, it is possible


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that Cry toxins could enter the fat body and affect the functions of Dorsal. On the

192

other hand, the Dorsal in the midgut juice may bind to Cry1Ab1 and form a

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Cry-Dorsal complex, leading to the sequestration of Cry toxin ingested by insects.

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Intriguingly, Crava et al. 22 reported a high transcriptional activation of AMPs

195

and lysozymes after treating S. exigua with Cry1Ca and Vip3Aa toxins, this was

196

previously described by the author as a mechanism to prevent septicemia caused by

197

commensal bacteria. However, it was reported by Paterson et al. 23 that continuous

198

activation of NF-κB after clearance of infected pathogens in mammals usually causes

199

damage and even septic injury. Kim et al. 24 showed that failure of negative cross-talk

200

that negatively regulates the AMP synthesis increased the lethality of bacterial

201

infection in Drosphila. Moreover, Broderick et al.

202

Lymantria dispar, inhibitory compounds that suppress the immune response delayed

203

B. thuringiensis-induced mortality and peptidoglycan fragments substituted for

204

Enterobacter in accelerating killing of antibiotic-treated larvae with B. thuringiensis.

205

The above evidences imply that Cry toxins might incite an over-reacted immune

206

response, in cooperation with other factor, which ultimately leads to targets’ death.

207

Notably, Dorsal can be converted form activator to repressor by Dorsal switch protein

208

(Dsp1),

209

The activation of IMD pathway also increases the expression level of certain AMPs,

210

which is utilized by the insect hosts to defend against gram’s negative pathogens. 15

211

The binding of Cry to Dorsal might hinder the converting process of Dorsal from an

212

activator to a repressor, leading to a malfunctioning of the negative regulation of the

213

Toll pathway; therefore, increasing the expression level of AMPs and this can be fatal

214

to the host insects.

26

25

found that, in gypsy moth

the latter plays pivotal role in the negative feedback of IMD pathway,

10


27

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Based on previous studies and our results, we proposed several possible

216

behaviors of Cry1Ab1 after ingestion by target insects: Firstly, after ingestion, Cry

217

interacts with Dorsal in the midgut juice, thereby leading to the sequestration of the

218

toxin (Fig.4); or, on the other hand, the interaction with Dorsal might facilitate the

219

binding of Cry to its receptors and leads to pore-formation (Fig.4). However, whether

220

the interaction of two proteins activates certain signaling pathways needs further

221

confirmation (Fig.4). Secondly, after entering into the fat body cells, the interaction

222

with Dorsal might lead to either the release of Dorsal from Cactus (Fig.4) or the

223

formation of Cry-Dorsal complex that ulteriorly block the original function of Dorsal.

224

Finally, Cry might serve as a Dorsal co-activator or co-repressor (Fig.4), thus will

225

either increase or decrease the expression level of Toll pathway-regulated AMPs, and

226

the two events are mutually exclusive. Considering previous studies in which an

227

increase in AMPs expression levels were observed, 22,25 Cry is more likely to serve as

228

a Dorsal co-activator, yet, it warrants further confirmation. On the other hand, the

229

interaction between Cry and Dorsal might prevent the conversion of Dorsal from an

230

activator to a repressor (Fig.4) by Dsp1 (Fig.4), leading to an increase in AMPs levels.

231

Crava et al. 22 found that after Cry1Ca treatment, almost all the transcripts coding for

232

AMPs and lysozymes were up-regulated, showing moderate (2–20 fold) to high

233

transcriptional activation (up than 20 fold); Zhong et al.28 found that co-expression of

234

Manduca

235

MsRelish2-RHD suppressed the activation of several AMP gene promoters, which

236

negatively regulates the host’s immune pathways. We thus speculate that Cry may

237

cause the activation of the immune pathways by interfering directly with the Toll

238

pathway or interfering with the negative feedback of the IMD pathway. (Fig.4). The

239

hypothetical modes of actions above may give rise to an over-firing immune response

sexta

Dorsal-Rel

Homology

Domain

11


(MsDorsal-RDH)

and


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which can be lethal to the insect hosts; still, further experiments are needed to confirm

241

our hypotheses.

242

Peroxidase-C was a Cry1Ab1-binding protein in the midgut juice of S. exigua Cry1Ab1-coupled

SepharoseTM

4B

beads

were

used

to

isolate

243

larvae.

244

Cry1Ab1-binding proteins from S. exigua midgut juice (Fig.5). One protein band

245

(Fig.5) was observed compared to control (Fig.5) in which CNBr-activated

246

SepharoseTM 4B coupled only with Tris buffer without Cry1Ab1 protein. The band

247

was analyzed by LC-MS/MS. Results indicated the band was peroxiredoxin-4

248

ortholog from Papilio machaon. BLAST using the sequence of P. machaon

249

peroxiredoxin-4 against S. exigua revealed another typical 2-Cys peroxiredoxin (PRX)

250

family protein: peroxidase-C (POX-C) (accession: KJ995804.1) which shared an

251

identity of 52.23% with the P. machaon peroxiredoxin-4. Sequence alignment showed

252

that two proteins were highly conserved in their thioredoxin domains, which were

253

residues 53-245 in PRX-4 and residue 2-191 in POX-C (Fig.6).

254

Notably, trypsin-like proteases identified in P. xylostella to be Cry1Ab1 binding

255

protein was not found in S. exigua. In the pore-forming mode, further processing of

256

the Cry toxic core after its interaction with membrane receptors is essential for toxin

257

oligomerization. 2 This led us to infer that the lack of certain proteases, in the midgut

258

juice of S. exigua, that further process the Cry toxic core might hinder the formation

259

of toxin oligomers, therefore reduces susceptibility of S. exigua to Cry1Ab1.

260

Molecular docking between Cry1Ab1 and two PRX proteins. 3-D structure of

261

PRX-4 and POX-C were constructed by homology modeling and were accessed using

262

RAMPAGE server, results showed over 95%, 95.4% and 97.9% amino acid residues

263

were in the favored region for Cry1Ab1, PRX-4 and POX-C respectively. Two

264

proteins were highly similar with respect to their tertiary structure (Fig.7). 12


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265

Molecular docking between Cry1Ab1 and two PRX proteins were performed

266

(Fig.7). Both PRX-4 and POX-C might interact with Cry1Ab1 by binding to the

267

groove formed by its three domains with all three domains in Cry1Ab1 participated in

268

the interaction (Fig.7). Analysis of the docking interfaces revealed several highly

269

conserved secondary structure elements that could bind to the Cry toxin (Fig.7).

270

Production of the reactive oxygen species (ROS) is one of the highly conserved

271

mechanisms with which insects defend against pathogens; 29 however, elevated ROS

272

due to oral infections caused the death of approximately half of the intestinal

273

epithelial cells, which is usually rescued by intestinal stem cell (ISC) proliferation. 30

274

As an antioxidant protein, peroxiredoxin-4 can reduce intracellular ROS, specifically

275

peroxides such as hydrogen peroxide, thereby influencing the signaling pathways

276

related to intracellular oxidation. In previous study on human cells, peroxiredoxin-4

277

was identified either as a cytoplasmic protein that attenuates NF-κB activation, 31 or as

278

a secretory protein, which serves as a proinflammatory cytokine, activating both

279

NF-κB and JNK. 32 In insects, JNK and JAK/STAT pathway were involved in the

280

repairing process after cell damage by ROS, 30 briefly: the JNK and Hippo-pathways

281

are activated in damaged enterocytes, where they produce Upd3, a ligand of Domeless

282

(Dome) that activates the JAK/STAT pathway in ISCs to proliferate and differentiate

283

into enterocytes. In Drosophila, the extracellular form of peroxiredoxin-4 is required

284

for activation of the JAK/STAT-mediated stress response. 33 Thus, the binding of

285

Cry1Ab1 to the extracellular peroxiredoxins may block its function in these signaling

286

processes.

287

Cry1Ab1 might cause insect’s death by interfering with peroxidase-related

288

immune responses. Peroxidases (POXs) are a sister group of cyclooxygenase genes

289

(COXs). COXs participate in the insect immune response by modulating the 13



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biosynthesis of prostaglandins (PGs) that mediate the insects’ hemocyte nodulation

291

and microaggregation reaction to bacterial infection.34 Park et al.

292

expression patterns and phylogeny of ten S. exigua POXs genes (SePOXs), among

293

which, two genes, namely SePOX-F and SePOX-H were found to be responsible for

294

PG production, however, the function of another eight POXs remained elusive.

295

Interestingly, we observed that in the study conducted by Park et al. 34 POX-C was

296

found to be expressed at a relatively higher level in the gut tissue than other tissue

297

studied, indicating that POX-C plays vital role in S. exiguagut immunity; however, the

298

expression level of S. exigua POX-C apparently decreased after treating the S. exigua

299

with bacteria,

300

expression level of peroxidases was responsible for cleaning up the excess ROS in the

301

hosts after the infection-driven increase of ROS. 29, 30, 35 A possible explanation is that

302

POX-C, like POX-F and –H, does not interact directly with ROS; additionally,

303

whether POX-C is involved in the JNK and JAK/STAT signaling pathways remained

304

elusive.

34

34

studied the

this was in contrast to previous studies in which the increased

305

Notably, typical 2-Cys peroxiredoxins were assigned with the name “2-Cys”

306

based on its two Cys residues that participate in the degradation of peroxides, the two

307

Cyc residues (Cys48 and Cys152) are conserved among all typical 2-Cys

308

peroxiredoxins. 35 Our molecular docking results indicated that Cys48 were involved

309

in the interaction between Cry1Ab1 with respect to both PRX-4 and POX-C (Fig.7);

310

Cys152 of PRX-4 also involved in the interaction (Fig.7); although Cys152 of POX-C

311

did not involve in the interaction, residues (E150-E151 and E154) resided at the two

312

sides of Cys152 participated in the binding with Cry1Ab1 (Fig.7), the steric hindrance

313

caused by the interaction can interfere with the residue’s chemical properties, this led

314

us to believe that the ingested Cry1Ab1 can block the peroxidase activity of these 14


Page 15 of 36


315

peroxidases that are

316

agents. 30

usually secreted into the extracellular spaces as anti-infection

317

Consider the two main functions of peroxiredoxins: the clearance of excess ROS

318

and the starting of repairing process by activating JNK and JAK/STAT pathways, Cry

319

binding to the peroxiredoxin can severely hinder the protective mechanisms triggered

320

by the cell damages caused by host’s immune responses, and this can cause more cell

321

damages upon infections. On the other hand, peroxiredoxin expression level is

322

elevated post-infections, 30, 35 the soluble proteins in the midgut juice can directly bind

323

to the ingested Cry1Ab1 leading to the neutralization of the toxin. To understand the

324

exact interaction patterns between Cry1Ab1 and POX-C, further work is warranted.

325

Conclusions and considerations. Insects’ resistance to Cry toxins has become a

326

major problem that hinders the application of Cry-based insecticides and transgenic

327

plants. Normally, the toxins are firstly ingested and processed into active form in the

328

midgut of insect larvae; however whether proteins in the midgut juice interfere with

329

the insecticidal process of Cry toxins is little known. In the present study, we

330

performed pull-down assay using a Cry1Ab1-coupled Sepharose beads to identify that

331

Dorsal and POX-C were the putative binding proteins in the midgut juice of P.

332

xylostella and S. exigua, respectively.

333

Dorsal is a nuclear factor that plays vital role in the insect immune response to

334

bacteria and fungi infections, thus the interaction of Cry1Ab1 with Dorsal can alter its

335

original functions. Based on previous studies and our results, we proposed possible

336

insecticidal mechanisms in which, Cry1Ab1 interferes with the Toll pathway by

337

bindind to Dorsal, thus leading to an over-firing immune response that speeds up the

338

killing process of Cry1Ab1.

15



339

POX-C and PRX-4 are members of the typical 2-cys peroxiredoxins. PRX-4 is

340

associated with the JNK and JAK/STAT pathways that mediate the repairing

341

processes after cell damage caused by ROS; POXs play important roles in the

342

regulation of ROS levels after infections. Molecular docking between Cry1Ab1 and

343

the two peroxiredoxins showed that Cry binding to PRX-4 and POX-C blocked the

344

two conserved Cys residues that usually participate in the degradation process of

345

peroxides; this can alter the peroxidase behavior of the peroxiredoxins. On the other

346

hand, the increased peroxiredoxin level post-infections might lead to the sequestration

347

of Cry1Ab1, thus weakening the toxicity of the toxin.

348

Our findings indicate that there exist Cry-binding proteins in the midgut juice of

349

insects which might interfere with the insecticidal process of the toxins. Also, we

350

speculate, based on our findings, that Cry1Ab1 might trigger over-firing immune

351

responses that can be lethal to respective insects. Notably, we found two trypsin-like

352

serine proteases that could bind to Cry1Ab1 in the midgut juice of P. xylostella but not

353

in that of S. exigua. The lack of certain proteases might cause insufficient processing

354

of Cry protoxins, hence making S. exigua insusceptible to Cry1Ab toxins. Future

355

works should address following questions: 1) validating the interactions between

356

Cry1Ab1 and its binding proteins; 2) determining the interaction patters between

357

Cry1Ab1 and its binding proteins; 3) determining the respective functions of

358

Cry-binding proteins in different insect species.

359

16


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ABRREVIATIONS USED Bt, Bacillus thuringiensis; BBMVs, brush border membrane vesicles; ALP, alkaline phosphatase; APN, Aminopeptidase; POX-C, peroxidase-C; ASP-1, aspartic protease-1; IPTG, isopropyl b-D-thiogalactoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC-MS/MS, liquid chromatography tandem mass spectrometry; PRX-4, Peroxiredoxin-4; AMPs, anti-microbial peptides; Dsp-1, dorsal switch protein; ROS, reactive oxygen species; ISC, intestinal stem cell; PGs, prostaglandins; Px, Plutella xylostella; Se, Spodoptera exigua. IMD-pathway, immune deficiency pathway; NF-κB, nuclear factor- κB; IκB, (inhibitor of κB); nuclear factor kappa-light-chain-enhancer of activated B cells; JNK, c-Jun N-terminal kinase; JAK/STAT, Janus kinase/Signal transducer and activator of transcription;;

Upd3,

unpaired-like

protein

3;

Dome,

domeless;

PGRP,

peptidoglycan recognition protein; Imd, immune deficiency protein; MyD88 myeloid differentiation primary response gene 88.

ACKNOWLEDGEMENTS This work was supported by the National Key Research and Development Program of China, and the Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (ZQN-YX205).

FUNDING SOURCES

17



This work was supported by National Key Research and Development Program of China and Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University (ZQN-YX205).

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12. Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Gallo Cassarino, T.; Bertoni, M.; Bordoli, L.; Schwede, T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014, 42, W252-W258. 13. Lovell, S.C.; Davis, I.W.; Arendall, W.B. 3rd; de Bakker, P.I.; Word, J.M.; Prisant, M.G.; Richardson, J. S.; Richardson, D. C. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins. 2003, 50(3), 437-450. 14. Pierce, B.G.; Wiehe, K.; Hwang, H.; Kim, B.H.; Vreven, T.; Weng, Z. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics. 2014, 30(12), 1771-1773. 15. Tanji, T.; Ip, Y.T. Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol. 2005, 26(4), 193-198. 16. Lemaitre, B.; Hoffmann, J. The host defense of Drosophila melanogaster. Annual review of immunology. 2007, 25, 697-743. 17. Vega-Cabrera, A.; Cancino-Rodezno, A.; Porta, H.; Pardo-Lopez, L. Aedes aegypti Mos20 cells internalizes cry toxins by endocytosis, and actin has a role in the defense against Cry11Aa toxin. Toxins. 2014, 6(2), 464-487. 18. Paula, D.P.; Souza, L.M.; Andow, D.A. Sequestration and Transfer of Cry Entomotoxin to the Eggs of a Predaceous Ladybird Beetle. PloS ONE. 2015, 10(12), e0144895.

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19. Zhang, G.F.; Wan, F.H.; Lövei, G.L.; Liu, W.X.; Guo, J.Y. Transmission of Bt Toxin to the Predator Propylaea japonica (Coleoptera: Coccinellidae) Through Its Aphid Prey Feeding on Transgenic Bt Cotton. Environ. Entomol. 2006, 35(1), 143-150. 20. Paula, D.P.; Andow, D.A.; Timbo, R.V.; Sujii, E.R.; Pires, C.S.; Fontes, E.M. Uptake and transfer of a Bt toxin by a Lepidoptera to its eggs and effects on its offspring. PloS one. 2014, 9(4), e95422. 21. Budatha, M.; Meur, G.; Dutta-Gupta, A. A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase. Biochem J. 2007, 405(2), 287-297. 22. Crava, C.M.; Jakubowska, A.K.; Escriche, B.; Herrero, S.; Bel, Y. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus. PloS one 2015, 10(5), e0125991. 23. Paterson, R.L.; Galley, H.F.; Dhillon, J.K; Webster, N.R. Increased nuclear factor kappa B activation in critically ill patients who die. Crit. Care Med. 2000, 28(4):1047-1051. 24. Kim, L.K; Choi, U.Y.; Cho, H.S.; Lee, J.S.; Lee, W.B.; Kim, J.; Jeong, K.; Shim, J.; Kim-Ha, J.; Kim, Y. J. Down-regulation of NF-kappaB target genes by the AP-1 and STAT complex during the innate immune response in Drosophila.

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25. Broderick, N.A.; Raffa, K.F.; Handelsman, J. Chemical modulators of the innate immune response alter gypsy moth larval susceptibility to Bacillus thuringiensis. BMC Microbiol. 2010, 10, 129.

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26. Valentine, S.A.; Chen, G.; Shandala, T.; Fernandez, J.; Mische, S.; Saint, R.; Courey, A. J. Dorsal-mediated repression requires the formation of a multiprotein repression complex at the ventral silencer. Mol Cell Biol. 1998, 18(11), 6584-6594. 27. Kleino, A.; Silverman, N. The Drosophila IMD pathway in the activation of the humoral immune response. Dev. Comp. Immunol. 2014, 42(1), 25-35. 28. Zhong, X.; Rao, X.J.; Yi, H.Y.; Lin, X.Y.; Huang, X.H.; Yu, X.Q. Co-expression of Dorsal and Rel2 Negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta. Sci. Rep. 2016, 6, 20654 . 29. Hillyer, J.F. Insect immunology and hematopoiesis. Dev. Comp. Immunol. 2016, 58:102-118. 30. Kuraishi, T.; Hori, A.; Kurata, S. Host-microbe interactions in the gut of Drosophila melanogaster. Front Physiol. 2013, 4, 375. 31. Jin, D.Y.; Chae, H.Z.; Rhee, S.G.; Jeang, K.T. Regulatory role for a novel human thioredoxin peroxidase in NF-kappaB activation. J. Biol. Chem. 1997, 272(49), 30952-30961. 32. Haridas, V.; Ni, J.; Meager, A.; Su, J.; Yu, G.L.; Zhai, Y.; Kyaw, H.; Akama, K. T.; Hu, J.; Van Eldik, L. J.; Aggarwal, B. B. TRANK, a novel cytokine that activates NF-kappa B and c-Jun N-terminal kinase. J. Immunol. 1998, 161(1), 1-6. 33. Radyuk, S.N.; Klichko, V.I.; Michalak, K.; Orr, W.C. The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signaling functions. FASEB J. 2013, 27(4), 1426-1438. 34. Park, J.; Stanley, D.; Kim, Y. Roles of peroxinectin in PGE2-mediated cellular immunity in Spodoptera exigua. PloS one. 2014, 9(9), e105717.

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35. Zhang, L.; Lu, Z. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori. Insect Mol. Biol. 2015, 24(2), 203-212.

FIGURE CAPTIONS Fig 1. SDS-PAGE analysis for the expression of pEASY- Cry1Ab1 in E. coli. M: pageruler prestained protein ladder；1：E. coli with pEASY vector；2：pellet of pEASY- Cry1Ab1 ; 3: supernant of pEASY- Cry1Ab1.

Fig 2. Purification of recombinant Cry1Ab1. M: pageruler prestained protein ladder；1：Denatured Cry1Ab1 inclusion bodies；2: Flow through after sample loading; 3: Flow through after 10th wash; 4: Eluent (Elution buffer containing 50mM imidazole); 5: Eluent (Elution buffer containing 150mM imidazole); 6: Eluent (Elution buffer containing 200mM imidazole).

Fig 3. Pull-down of Cry1Ab1-binding proteins from midgut juice of P. xylostella. M ： marker ； 1, Cry1Ab1-binding protein (1st pull-down) ； 2, Cry1Ab1-binding protein (2nd pull-down) ； 3, Cry1Ab1-binding protein (3rd pull-down)；4, control. Red arrow indicates protein bands of two trypsin-like serine proteases and Dorsal; Blue arrow indicates protein bands of putative zinc finger protein, putative hemodomain transcription facto and an uncharacterized protein.

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Fig 4. Schematic representation of a possible insecticidal mechanism of Cry1Ab1. a) Three possible behaviors after Cry1Ab1 ingestion and interaction with Dorsal in the midgut juice: 1. Sequestration by Dorsal, 2. starting certain signaling pathways, 3. Dorsal facilitates the binding to receptors that lead to pore-formation; b) Possible interaction patterns between Cry1Ab1 and Dorsal: 1. Cry1Ab1 binds to Dorsal therefore releasing Dorsal previously trapped by Cactus, 2. Cry1Ab1 binds to Dorsal forming a Cry-Dorsal complex that changes the behavior of Dorsal; c) Possible insecticidal mechanisms triggered by Cry-Dorsal: 1.Cry1Ab1 binds to Dorsal as co-activator that facilitate the transcription of related AMPs, 2. Cry1Ab1 binds to Dorsal as co-repressor that hinder the transcription factor function of Dorsal, 3. Cry1Ab1 raises AMPs levels by directly interacting with the negative feedback loops of the IMD pathway, 4. Cry1Ab1 raises AMPs levels by preventing Dorsal from being converted to repressor by Dsp1; d) IMD and Toll pathways in the absence of Cry toxin: 1. IMD pathway, upon Gram-negative bacterial infection, PGRP (peptidoglycan recognition protein) receptor activates Imd (immune deficiency) and Relish sequentially, therefore starting AMPs transcription, 2.Toll pathway, upon fungi or Gram-positive bacterial infection, Toll receptor activates downstream adaptor protein MyD88 (myeloid differentiation primary response gene 88), which leads to the degradation of Cactus, therefore releasing Dorsal and starting AMPs transcription. Grey circle: Cry1Ab1; Balck circle: sequestrated Cry1Ab1; Green circle: Active-form of Dorsal; Red circle: Repressor-form of Dorsal; Blue box:

25



Cactus; Orange oval: Dsp1; Pink circle: Relish; Dotted circles: receptors and messengers; Red solid line: negative feedbacks of IMD pathway; Dotted line: speculated processes; Solid line: confirmed processes; Magenta solid lines: Plasma membrane; Brown dotted line: Nuclear membrane.

Fig 5. Pull-down of Cry1Ab1-binding proteins from midgut juice of S. exigua. M：marker；1, Cry1Ab1-binding protein (1st pull-down)；2, Cry1Ab1-binding protein (2nd pull-down)；3, Cry1Ab1-binding protein (3rd pull-down)；4, control. Red arrow indicates the protein band of Peroxiredoxin-4.

Fig 6. Sequence alignment between PRX-4 and POX-C. Dark blue: highly conserved; Light blue: moderately conserved

Fig 7. Molecular dockings between Cry1Ab1 and two proteins. a) binding pattern between Cry1Ab1 (aquamarine) and POX-C (magenta); b) binding interface (magenta) in POX-C; c) binding pattern between Cry1Ab1(aquamarine) and PRX-4 (prasinous); d) binding interface (green) in PRX-4

26


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TABLES Table 1. Cry1Ab-binding proteins in the midgut juice of P. xylostella and S. exigua Speciesa

Px

Bandb

Red

Masco

Blast

Query

t score

score

cover

155

471

93%

Identity

Accession No.

Description

100%

gi|347810670

Trypsin-like serine proteinase 2 OS=Plutella xylostella

Px

Red

15

374

77%

98%

gi|347810668

Trypsin-like serine proteinase 1 OS=Plutella xylostella

Px

Red

13

848

100%

100%

gi|703890272

Dorsal OS=Plutella xylostella

Px

Blue

44

2728

100%

100%

gi|357614269|EHJ6

Putative Zinc finger protein

8997.1

294 OS=Danaus plexippus

gi|914571398|KOB

Putative

74527.1

transcription factor (Fragment)

Px

Blue

24

1251

100%

100%

homeodomain

OS=Operophtera brumata Px

Blue

23

3995

100%

96%

XP_012549385.1

Uncharacterized

protein

OS=Bombyx mori Se

Red

31

508

100%

100%

gi|943970231

Peroxiredoxin-4 machaon

a

Px: Plutella xylostella, Se: Spodoptera exigua 27


OS=Papilio


b

Red: band indicated by red arrow in corresponding SDS-PAGE profile, Blue:

band indicated by blue arrow in corresponding SDS-PAGE profile.

28


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FIGURE GRAPHICS

Fig 1. SDS-PAGE analysis for the expression of pEASY- Cry1Ab1, pEASY-AG and pEASY-AP in E. coli.

29



Fig 2. Purification of recombinant Cry1Ab1.

30


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Fig 3. Pull-down of Cry1Ab1-binding proteins from midgut juice of P. xylostella.

31



Fig 4. Schematic representation of a possible insecticidal mechanism of Cry1Ab1.

32


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Fig 5. Pull-down of Cry1Ab1-binding proteins from midgut juice of S. exigua.

33



Fig 6. Sequence alignment between PRX-4 and POX-C.

34


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Fig7 . Molecular dockings between Cry1Ab1 and two proteins.

35



TOC GRAPHIC

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Possible Insecticidal Mechanisms Mediated by ... - ACS Publications

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