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Screening and functional analyses of Nilaparvata lugens salivary proteome Hai-Jian Huang, Cheng-Wen Liu, Xiao-Hui Huang, Xiang Zhou, Ji-Chong Zhuo, Chuan-Xi Zhang, and Yan-Yuan Bao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00086 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 6, 2016

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Journal of Proteome Research

Screening and functional analyses of Nilaparvata lugens salivary proteome

Hai-Jian Huang, Cheng-Wen Liu, Xiao-Hui Huang, Xiang Zhou, Ji-Chong Zhuo, Chuan-Xi Zhang, Yan-Yuan Bao*

State Key Laboratory of Rice Biology and Ministry of Agriculture Key Laboratory of Agricultural Entomology, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

* Corresponding author. Tel: +86-571-88982995; Fax: +86-571-88982991 E-mail address: [email protected] (Y.-Y. Bao)

E-mail addresses for all authors Yan-Yuan Bao: [email protected] Hai-Jian Huang: [email protected] Cheng-Wen Liu: [email protected] Xiao-Hui Huang: [email protected] Xiang Zhou: [email protected] Ji-Chong Zhuo: [email protected] Chuan-Xi Zhang: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva, displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath; while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests. Key words: Planthopper; salivary protein; proteome; salivap; annexin-like; CA; RNA interference 2


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INTRODUCTION In plant sap-sucking insects, saliva plays a crucial role in determining the compatibility between the insects and their host plant1. The saliva contains bioactive proteins that have an array of functions from countering plant defense mechanisms to digestion of nutrients, and thus, allowing insects to feed and survive successfully. The continuous secretion of saliva, while feeding, is an important process that conditions feeding sites and enables sap sucking for hours to days 2. Most phloem-feeding hemipteran insects during the feeding process secrete two types of saliva, gelling and watery. On release into the plant tissues, the gelling saliva coagulates and forms a salivary sheath around the stylets, which provides mechanical stability and lubrication and protects the insect against chemical defenses. The watery saliva is secreted into the host tissue, but mostly in the phloem, and is considered to contain bioactive components involved in the suppression or induction of plant defense responses

2-4

. Proteome analyses of the salivary components have

already been performed in phytophagous sap-sucking insects, including several aphid species 2, 5-10 and the green rice leafhopper, Nephotettix cincticeps

11

. However, studies on the salivary

components and functions of monophagous sap-sucking insect species are limited. The brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae) is a monophagous sap-sucking insect pest that exclusively depends on rice sap. N. lugens causes serious damage to rice by sucking phloem sap and transmitting plant viruses 12. These factors can cause severe yield reductions and significant economic losses in rice crop throughout East Asian countries

13

. Like

most phloem-feeding hemipteran insects, N. lugens secretes gelling and watery saliva from its salivary glands. The transcriptome and proteome of salivary glands in N. lugens have been analyzed

14, 15

. The functions of salivary gland extract and several secretory proteins, i.e. a 3



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catalase-like protein and a salivary sheath protein have been characterized16-18. More bioactive components in N. lugens saliva need to be discovered. To better understand the biological characteristics of the secreted saliva in the interactions between N. lugens and the rice plant, we were interested in identifying the protein components in N. lugens watery and gelling saliva. In our previous work, we completed the whole-genome sequencing of N. lugens and obtained the gene annotation19. The N. lugens genome is the first characterized genome of a monophagous sap-sucking arthropod herbivore. We performed RNA-Seq transcriptome sequencing, which provided detailed gene expression information regarding the developmental stages and tissue specificity in N. lugens

20-23

. The availability of genomic and transcriptomic datasets can be

exploited by us in the proteomic analysis of salivary proteins in N. lugens. In this study, to identify secreted salivary proteins from artificial diets, we conducted proteomic analyses combined with the genomic and transcriptomic analyses. Our results revealed that some salivary proteins were species-specific due to their unique identification in N. lugens and not in other insect species. Some proteins, i.e., a group of annexin-like proteins, were first detected in insect saliva. A further functional analysis by RNAi revealed the importance of three salivary proteins in the feeding process and survival of N. lugens. The identification and the functional analysis of these salivary proteins provide possibility to understand some aspects of plant-insect molecular interaction mechanisms and identify potential targets for pest management.

MATERIALS AND METHODS Insects The N. lugens populations used for collecting secreted saliva in this work were originally collected 4


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from a rice field in the Huajiachi Campus of Zhejiang University, Hangzhou, China, in 2008. The insects were reared at 26 ± 0.5°C with 50 ± 5% humidity on rice seedlings (Oryza sativa strain Xiushui 134) under a 16:8 h light:dark photoperiod as previously described 19. Collection and concentration of watery saliva protein The fifth instar N. lugens nymphs were transferred from the rice seedlings to sterile diets with 2.5% sucrose and reared for 24 h. The collection of saliva for each biological repeat used 8,000 nymphs feeding on dietary sucrose held between two layers of ParafilmTM M Laboratory Film (Neenah, WI, USA). The sucrose diet was prepared under aseptic conditions and filtered through a 0.22 µm syringe filter (Millipore, MA, USA). The liquid was collected from the space between the two layers of Parafilm with a pipette 24 h after feeding on dietary sucrose and was centrifuged at 7,000 g at 4°C for 30 min. The supernatant was ultrafiltered with a 3-kDa molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device (Millipore). One milliliter of the ultrafiltered sample was concentrated by adding five times the volume of 10% trichloroacetic acid-acetone solution at –20°C for overnight. The pellet was added to five times volume of cold-acetone and centrifuged at 7,000 g at 4°C for 30 min. The protein pellet was solubilized in 200 µl SDT buffer (4% sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO, USA), 100 mM dithiothreitol (DTT; Sigma), 150 mM Tris-HCl pH 8.0) and incubated in hot water for 15 min, then centrifuged at 14,000 g for 45 min. In-solution trypsin digestion Protein digestion was performed according to a filter-aided sample preparation (FASP) method 24. Briefly, 40 µl of protein sample was added to 100 mM DTT and boiled for 5 min. DTT and other low-molecular-weight components were removed using 200 µl of UA buffer (8 M Urea, 150 mM 5



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Tris-HCl pH 8.0) by repeated ultrafiltration using 3-kD filtration units (Millipore) at 14,000 g for 15 min three times. Proteins were alkylated with 100 µl of 0.05 M iodoacetamide (IAA; Sigma) in UA buffer by vortexing for 1 min and incubated at room temperature in the dark for 30 min. The filter was washed with 100 µl of UA buffer at 14,000 g for 10 min twice and then 100 µl of 25 mM NH4HCO3 (Sigma) twice. Finally, the protein suspension was digested with 4 µg trypsin (Sigma) in 40 µl of NH4HCO3 buffer by vortexing for 1 min and incubated at 37°C overnight. The digested peptides were collected by concentrating at 14,000 g for 10 min and were quantified at OD280. Liquid chromatography−mass spectrometry/mass spectrometry of watery saliva protein The shotgun analysis was conducted on an Easy nLC (Thermo Fisher Scientific, MA, USA) coupled with a Q Exactive mass spectrometer (Thermo Finnigan, CA, USA). Peptides were desalted on C18 Cartridges (EmporeTM C18-High Performance Extraction Disk Cartridge 4215, standard density, 3M). The samples were concentrated by vacuum centrifugation and reconstituted in 10 µl of 0.1% (v/v) trifluoroacetic acid. Each fraction (6 µl) was injected for nanoLC-MS/MS analysis. The peptide mixture (5 µg) was loaded onto the C18-reversed phase column (Thermo Scientific Easy Column, 2 cm long, 100 µm inner diameter, 5 µm resin) in buffer A (0.1% formic acid) and separated on an analytical column (RP-C18, 75 µm × 100 mm, 3 µm resin Thermo Scientific Easy Column) with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 250 nl/min controlled by IntelliFlow technology over 140 min. Separated MS data was analyzed using a Q Exactive mass spectrometer by dynamically choosing the 10 most intense ions from one full mass scan (300–1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. Determination of the target value is based on predictive automatic gain control (pAGC). Dynamic exclusion duration was 60 s. Survey scans were acquired at a resolution of 6


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70,000 at m/z 200 and the resolution for HCD spectra was set to 17,500 at m/z 200. Normalized collision energy was 27 eV and the under fill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled. Collection and preparation of gelling saliva protein and LC-MS/MS The fifth instar N. lugens nymphs were fed with 2.5% sucrose for 24 h as described above. The salivary sheaths were carefully separated from Parafilm using forceps under a stereomicroscope (Leica S8AP0, Wetzlar, Germany). Each collection of salivary sheaths was from at least 6,000 nymphs.

The

collected

salivary

sheaths

were

solubilized

in

lysis

buffer

(4%

3-[(3-cholamido-propyl)-dimethylammonio]-1-propanesulfonate (Yeasen, Shanghai, China), 2% SDS and 2% DTT) and boiled for 10 min. The protein sample was denatured in 9 M urea at room temperature for 1 h, followed by precipitating using a trichloroacetic acid protein precipitation kit (Sangon, Shanghai, China). The sample was concentrated with a 3-kDa filtration unit and alkylated with 200 mM IAA. The protein sample was digested with trypsin in 50 mM NH4HCO3 buffer overnight at 37°C. LC-MS/MS analysis was performed as previously described 25. Briefly, the peptide mixture (20 µl) was loaded onto the trap column at a flow rate of 10 µl/min by Thermo Scientific Easy nanoLC 1000 (Thermo Fisher Scientific, MA, USA). After trap equilibration, the samples were eluted with a linear gradient of buffer A and B at a flow rate of 250 nl/min as described above. The chromatographic system includes a trapping column (75 µm×2 cm, nanoviper, C18, 3 µM, 100 Å) and an analytical column (50 µm×15 cm, nanoviper, C18, 2 µM, 100 Å). Separated MS data was analyzed using Thermo LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific, MA, USA) equipped Nanospray Flex ionization source and FTMS (Fourier transform 7



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ion cyclotron resonance mass analyzer) combined with Thermo LTQ-Orbitrap Elite equipped Ion Trap analyzer. The top 20 ions were chosen from a full mass scan (300–2000 m/z) by collision induced decomposition (1.0m/z isolation width, 35% collision energy, 0.25 activation Q, 10 ms activation time). Dynamic exclusion duration was 60 s. Survey scans for MS1 were acquired at a resolution of 30,000 at m/z 400. Bioinformatic analysis MS/MS spectra were searched using MASCOT engine (Matrix Science, London, UK; version 2.2) against N. lugens genomic (http://www.ncbi.nlm.nih.gov/, BioProject PRJNA177647, 27,571 coding protein sequences) and transcriptomic databases (http://www.ncbi.nlm.nih.gov/sra, accession number SRX023419, 21,908 coding protein sequences). Identified N. lugens sequences were validated using BLASTX to search for similar sequences in National Center for Biotechnology Information (NCBI) protein database with a significance cut-off of E-value < 10-5. For protein identification, the following options were used: (i) Peptide mass tolerance: 20 ppm; (ii) MS/MS tolerance: 0.1 Da; (iii) Enzyme: trypsin; (iv) Missed cleavage: 2; (v) Fixed modification: carbamidomethyl (C); (vi) Variable modification: oxidation (M); (vii) Ion score: >20. An automatic decoy database search was performed with false discovery rate (FDR) ≤0.01. The peptides with high peptide confidence (p50% are shown on each node of the tree. The GenBank accession numbers for the sequences are as follows: N. lugens annexin B9 (KU366704); Acyrthosiphon pisum annexin-B9 (XP_001949978); Bombyx mori annexin-B9 (XP_012550244); Papilio polytes 32


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annexin-B9 (XP_013147581); Drosophila melanogaster annexin-B9 (AAG12161); Anopheles darlingi annexin-B9 (ETN60044); N. lugens annexin-B10 (KU366705); Diaphorina citri annexin-B10 (XP_008470433); B. mori annexin-B10 (XP_012553385); P. polytes annexin-B10 (XP_013145711); D. melanogaster annexin-B10 (NP_476615); A. darlingi annexin-B10 (ETN63497); N. lugens annexin-like1 (KU365920); N. lugens annexin-like2 (KU365921); N. lugens annexin-like3 (KU365926); N. lugens annexin-like4 (KU365941) and N. lugens annexin-like5 (KU365947). N.l, N. lugens; A.p, A. pisum; B.m, B. mori; P.p, P. polytes; D.m, D. melanogaster; A.d, A. darling; D.c, D. citri.

Figure 2. Gene ontology classification of the watery saliva components. N. lugens watery saliva proteins are summarized in three main categories: biological process, molecular function and cellular component. The Venn diagram (http://bioinfogp.cnb.csic.es/tools/venny/) indicates the number of salivary proteins identified in watery and/or gelling saliva in N. lugens.

Figure 3. Tissue-specific expression (in fold) of N. lugens genes encoding salivary proteins. Total RNA was extracted from the salivary gland, gut, fat body, ovary, testis and the remaining carcass of N. lugens individuals (n=20–80). Total RNA from the whole insect body (n=8) was used as reference RNA. First-strand cDNA was analyzed in each quantitative real-time PCR reaction. The relative transcript levels of each gene in the tested tissues and whole insect body were normalized using N. lugens 18s rRNA threshold cycle (Ct) values. Three biological replications (mean ± SD) were carried out for each gene based on independent samples and the ∆∆Ct method was used to measure the relative transcript levels in each tissue. Results of triplicate experiments are shown 33



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with the standard deviations. The asterisk (*) indicates statistical significance at p < 0.01, different from each other tissue and whole insect body. Sg, salivary gland; Fb, fat body; Ov, ovary; Te, testis; Ca, the remaining carcass; Wh, whole insect body.

Figure 4. Effects of dsRNA interference on the survival of N. lugens. The third instar nymphs were injected with specific dsRNA and were observed for phenotypic variations at 24 h intervals. (A) injection with double-stranded RNA of mucin or salivap genes; (B) injection with double-stranded RNA of ANX-like genes; (C) injection with double-stranded RNA of genes encoding several enzymes. Individuals treated with dsGFP were used as a control. The survival rates were calculated from three biological replicates (mean ± SD). Each treatment n=50 nymphs. P < 0.05 was considered statistically significant (*), different from dsGFP treatment. The gene expression variations (in %) of salivap-3, ANX-like5 and CA at 5 d p.i. in N. lugens were analyzed by quantitative real-time PCR as described in Figure 3 and shown in the right panel (D, E, F). N indicates no treatment control.

Figure 5. Morphological observations of the salivary sheath in dsRNA-treated N. lugens. (A) dsGFP; (B) dssalivap-3; (C) dsANX-like5; (D) dsCA. The third instar N. lugens nymphs were injected with double-stranded target RNA. The nymphs at 5 d p.i. were fed with dietary sucrose for 24 h. Then the salivary sheaths were collected and observed under SEM.

Figure 6. Electrical penetration graph (EPG) monitoring of dsRNA-treated N. lugens nymphs on rice plants. (A) dssalivap-3 treatment; (B) dsANX-like5 treatment; (C) dsCA treatment. The third 34


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instar N. lugens nymphs were injected with double-stranded target RNA. The nymphs at 5 d p.i. were used in the experiment. N1, penetration initiation; N2, salivation and stylet movement; N3, extracellular activity near the phloem; N4, phloem sap ingestion; N5, xylem sap ingestion; np: non-penetration. All EPG recordings were performed for 8–10 h with at least ten replicates (mean ± SD) for each treatment. P < 0.05 was considered statistically significant (*), from dsGFP treatment.

Figure 7. Investigation of honeydew excretion in dsRNA-treated N. lugens. The 5th instar N. lugens nymphs (10 nymphs as a group) were wrapped in a Parafilm sachet for 24 hours. The excreted honeydew was collected and weighed. At least 20 replicates (mean ± SD) were performed for each treatment. P < 0.05 was considered statistically significant (*), different from dsGFP treatment.

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Table 1. N. lugens watery saliva proteins #

Protein

GenBank ID

Unique PepCount

Pep Count

Cover Percent

MW

PI

Signal P

Best match

Similaritya

E-valueb

1

carboxylesterase

AAG40239

18

46

37.11%

59987.15

5.51

Yes

Nilaparvata lugens

100%

0.0

2

annexin-like 1 (ANX-like 1)

KU365920

14

39

38.20%

42023.38

5.48

Yes

Lucilia cuprina

40%

5e-09

3


KU365921

13

30

43.22%

39042.50

5.15

Yes

Anopheles darlingi

48%

3e-23

4

venom dipeptidyl peptidase 4-like (VDDP-4 like)

KU365922

12

21

18.98%

82615.15

5.38

Yes

Cerapachys biroi

52%

2e-112

5

salivap-1

KU365923

11

14

31.16%

32625.29

4.84

Yes

No

6

alpha-N-acetylgalactosaminidase (alpha-GalNAc)

KU365924

9

21

22.33%

46965.76

5.00

Yes

Acyrthosiphon pisum

76%

0.0

7

arginine kinase

KU365925

9

10

24.72%

40027.91

5.69

No

Nephotettix cincticeps

97%

0.0

8


KU365926

7

14

30.30%

39376.90

5.30

Yes

Atta cephalotes

48%

3e-14

9

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

KU365927

6

10

17.17%

35454.09

8.19

No

Laodelphax striatella

99%

0.0

10

regucalcin-like

KU365928

6

9

16.78%

33013.76

6.37

Yes

Plutella xylostella

58%

3e-60

11

actin

KU365929

6

7

16.49%

41847.48

5.30

No

Drosophila melanogaster

99%

0.0

12

peritrophin-like protein1

KU365930

5

19

28.26%

25604.49

5.63

Yes

Riptortus pedestris

51%

3e-27

13

enolase

AHB33499

5

8

14.75%

47156.34

6.34

No

Nilaparvata lugens

100%

0.0

14

gamma-glutamyltranspeptidase 1 (GGT 1)

KU365931

5

6

7.08%

58149.28

5.92

Yes

Tribolium castaneum

58%

3e-109

15

salivap-2

KU365932

4

11

30.77%

16487.94

10.06

Yes

No

16

carbonic anhydrase (CA)

KU365933

4

9

15.19%

26976.63

8.53

Yes

Stomoxys calcitrans

54%

2e-34

17

peritrophin-like protein2

KU365934

4

8

15.61%

27225.13

5.17

Yes

Riptortus pedestris

48%

5e-29

18

calmodulin

KU365935

4

4

26.85%

16810.4

4.09

No

Drosophila melanogaster

100%

1e-100

36


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19

histone H2B

KU365936

4

4

21.25%

17657.41

10.41

No

Oryza brachyantha

100%

6e-81

20

salivap-3

KU365937

3

15

67.27%

4931.84

9.74

Yes

Nilaparvata lugens

100%

0.0

21

mucin-like protein

BAP87097

3

13

7.80%

78937.51

9.04

Yes

Nilaparvata lugens

99%

0.0

22

capsid protein

AIY53984

3

8

12.44%

24264.04

5.89

No

Himetobi P virus

100%

0.0

23

heat shock cognate protein 70

ADE34170

3

5

4.59%

71447.92

5.47

No

Nilaparvata lugens

100%

0.0

24

mitochondrial ATP synthase alpha subunit

KU365938

3

5

4.72%

59772.29

9.13

No

Acyrthosiphon pisum

99%

0.0

25

lysosomal acid phosphatase (LAP)

KU365939

3

4

6.23%

38842.38

9.27

Yes

Pediculus humanus corporis

63%

2e-77

26

maltase 2-like

KU365940

3

4

3.68%

66216.17

5.38

Yes

Acyrthosiphon pisum

70%

0.0

27


KU365941

3

4

9.44%

39978.77

6.81

Yes

Xiphophorus maculatus

44%

1e-13

28

14-3-3 zeta protein

KU365942

3

4

9.72%

28130.15

4.78

No

Riptortus pedestris

99%

1e-152

29

elongation factor 1-alpha

KU365943

3

4

19.92%

26230.88

5.99

No

Nilaparvata lugens

98%

6e-171

30

venom dipeptidyl peptidase (VDDP)

KU365944

3

4

4.57%

81954.79

6.19

Yes

Cerapachys biroi

56%

3e-159

31

phosphatidylcholine-sterol acyltransferase (LCAT)

KU365945

3

3

7.44%

45769.47

5.44

Yes

Tribolium castaneum

66%

1e-117

32

lysosomal alpha-mannosidase-like

KU365946

3

3

2.25%

116770.1

5.93

Yes

Acyrthosiphon pisum

70%

0.0

33


KU365947

3

3

10.07%

43364.4

5.19

Yes

Musca domestica

43%

1e-15

34

enolase 2

KU365948

2

6

25.93%

11713.2

6.78

No

Nicotiana sylvestris

97%

3e-66

35

histone H1

KU365949

2

5

14.88%

13512.61

11.45

No

Culex quinquefasciatus

95%

4e-48

36

transformation/transcription domain-associated protein (TRRAP)

KU365950

2

4

1.17%

107235.3

9.07

No

Megachile rotundata

82%

0.0

37

cathepsin B-like protease

CAC87118

2

3

4.90%

38982.81

7.84

Yes

Nilaparvata lugens

100%

0.0

38

aminopeptidase N-like

KU365951

2

3

2.50%

101638.6

5.89

Yes

Athalia rosae

55%

1e-164

39

14-3-3 protein epsilon

KU365952

2

2

6.87%

29817.14

4.69

No

Solenopsis invicta

98%

2e-174

37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 38 of 48

40

heat shock protein 60

KU365953

2

2

2.99%

60185.64

5.44

No

Acromyrmex echinatior

93%

0.0

41

myoneurin-like

KU365954

2

2

8.24%

28723.58

5.19

No

Bombus impatiens

39%

9e-11

42

trypsin-like protease

AID60343

2

2

6.29%

35311.58

6.55

Yes

Nilaparvata lugens

99%

0.0

43

methylcrotonoyl-CoA carboxylase subunit alpha

KU365955

2

2

7.34%

19101.83

9.23

No

Nasonia vitripennis

85%

6e-131

44

phosphatidylinositol-specific phospholipase C X domain-containing protein 1 (PLCXD1)

KU365956

2

2

4.56%

46104.97

5.38

Yes

Pogonomyrmex barbatus

48%

1e-36

45

tubulin alpha-2

KU365957

2

2

5.56%

50040.11

5.01

No

Laodelphax striatella

100%

0.0

46

calreticulin

KU365958

2

2

4.21%

46507.46

4.47

Yes

Apis mellifera

85%

2e-173

47

aldose 1-epimerase-like

KU365959

2

2

4.40%

37874.67

6.01

Yes

Orussusabietinus

64%

2e-101

48

leucyl aminopeptidase

KU365960

2

2

4.17%

54151.87

6.46

No

Riptortus pedestris

73%

0.0

49

plasma kallikrein-like (PKL)

AID60327

2

2

7.97%

27285.16

8.61

Yes

Nilaparvata lugens

100%

0.0

50

protein disulfide-isomerase

KU365961

2

2

3.39%

56774.62

4.71

Yes

Zootermopsis nevadensis

79%

0.0

MS/MS spectra were searched using MASCOT engine (Matrix Science, London, UK; version 2.2) against N. lugens genomic (http://www.ncbi.nlm.nih.gov/, BioProject PRJNA177647) and transcriptomic databases (http://www.ncbi.nlm.nih.gov/sra, accession number SRX023419). The sequences were validated using BLASTX in NCBI protein database with a significance cut-off of E-value < 10-5. Total pep count is the number of peptides identified by MS/MS. Unique is the number of unique pep count. Sequence coverage (%) means the number of amino acids spanned by the assigned peptides divided by the sequence length. The peptides identified by MS/MS with statistically significant ion score (P

Screening and Functional Analyses of Nilaparvata ... - ACS Publications

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