An Ovarian Protein Involved in Passive Avoidance of an

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Cite This: J. Proteome Res. 2019, 18, 2695−2705

An Ovarian Protein Involved in Passive Avoidance of an Endoparasitoid To Evade Its Host Immune Response Ziwen Teng, Huizi Wu, Xinhai Ye, Shijiao Xiong, Gang Xu, Fang Wang, Qi Fang, and Gongyin Ye* State Key Laboratory of Rice Biology & Ministry of Agricultural and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

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ABSTRACT: Through a combination of transcriptomic and proteomic analyses, we identified 817 secreted ovarian proteins from an endoparasitoid wasp, Cotesia chilonis, of which five proteins are probably involved in passive evasion. The results of an encapsulation assay revealed that one of these passive evasion-associated proteins (Crp32B), a homologue of a 32-kDa protein (Crp32) from C. rubecula, could protect resin beads from being encapsulated by host hemocytes in a dose-dependent manner. Crp32B is transcribed in ovarian cells, nurse cells, follicular cells, and oocytes, and the protein is located throughout the ovary and on the egg surface. Moreover, Crp32B has antigenic similarity to several host components. These results indicate that C. chilonis may use molecular mimicry as a mechanism to avoid host cellular immune response. KEYWORDS: parasitoid wasp, ovarian protein, passive strategy, Crp32, encapsulation, mimicry

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immune responses.16 In Cotesia kariyai, an immunoevasive protein (IEP) from the ovary has been reported to protect parasitoid eggs from host cellular reaction.17−19 Similarly, C. rubecula eggs are protected by a 32-kDa surface protein (Crp32). However, the molecular mechanism of Crp32mediated protection is not well-known.20 In contrast, the passive avoidance of host encapsulation in other parasitoids is mediated by molecules of extraembryonic membrane. A 97-kDa transmembrane hemomucin homologue from M. cingulum containing 51 potential O-glycosylation sites has been found to protect embryos from being encapsulated by their host, an activity in which its sugar chains play an important role.21 A polyembryonic encyrtid Copidosoma f loridanum, remains enveloped by an extraembryonic membrane throughout development, which is required for protection from the host immune system.22 Cotesia chilonis (Hymenoptera: Braconidae) is an obligate larval endoparasitoid that effectively regulates the population density of the striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae), one of the most economically important rice pests in Asia, Northern Africa, and Southern Europe.23,24 In our previous studies, C. chilonis was observed to use a combination of active and passive strategies to escape from host cellular immune responses.25 Additionally, we identified two venom-associated passive avoidance-related proteins, IEP2A (Accession No. KU663635) and IEP-2B (Accession No. KU663636) through a combination of transcriptomic and

INTRODUCTION To successfully develop in the host hemocoel, endoparasitoids have evolved diverse strategies in different parasitoid−host systems that can be categorized as “active” or “passive”.1,2 For the “active” strategy, parasitism factors include venoms,3 polydnaviruses (PDVs),4 teratocytes,5 virus-like particles (VLPs),6 and ovarian proteins7 that can suppress host immune responses. Numerous intensive investigations have been performed to identify and characterize PDV gene products,8,9 venom proteins,3,10 teratocyte secretory products,5,11 and VLP components6,12 by classical approaches and high-throughput sequencing. However, compared to other parasitism factors, ovarian proteins have been paid little attention. In contrast to “active” strategies, endoparasitoids also have evolved ‘‘passive strategies” in which endoparasitoids develop in the host hemocoel but passively avoid encapsulation by the host because of their progeny surface features. The results showed that the parasitoid progeny were not recognized as nonself by host hemocytes, resulting in host hemocytes being unable to attach to the progeny. In two braconid wasps, Toxoneuron nigriceps and Macrocentrus cingulum, a surface fibrous layer of the eggs has been shown to play an important role in protecting eggs from host immune responses.13,14 In the ichneumonid wasp V. canescens, egg-covering VLP proteins were shown to be antigenically related to a host 42 kDa protein, suggesting that the particles may not be recognized as a foreign substance.15 In addition to VLPs, Venturia hemomucin, a component of the mucinous layer on the egg and larval surface, can form a complex with host lipophorin and other hemolymph components. This protective mucinous layer protects parasitoid eggs from host © 2019 American Chemical Society

Received: October 17, 2018 Published: June 6, 2019 2695

DOI: 10.1021/acs.jproteome.8b00824 J. Proteome Res. 2019, 18, 2695−2705

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Journal of Proteome Research proteomic analyses.10 In the current study, we identify secreted ovarian proteins by C. chilonis through a combination of transcriptomic and proteomic approaches, resulting in the identification of five proteins that are likely involved in passive evasion. Moreover, we present evidence that Crp32B, one of the identified passive avoidance-related proteins, may be used as a molecular mimic involved to promote the passive evasion of C. chilonis eggs from the host immune response. Our results will provide insight into a more comprehensive understanding of the secreted ovarian components and the functions of ovarian proteins associated with passive strategy to evade host immune response for parasitoid wasps.

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positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300−1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. The settings used were as follows: gain control target: 1 × 106; maximum inject time: 50 ms; dynamic exclusion duration: 60.0 s; resolution of survey scans acquired: 70 000 at 200 m/z; resolution for HCD spectra: 17 500 at 200 m/z; isolation width: 2 m/z; normalized collision energy: 30 eV; and underfill ratio: 0.1%. The MS data were analyzed using MaxQuant version 1.5.3.17 (Max Planck Institute of Biochemistry in Martinsried, Germany)27 and were searched against the translated FPOvary and FAOvary transcriptomes. We set an initial search at a precursor mass window of 6 ppm, and then performed an enzymatic cleavage rule of trypsin. The following parameters were set: MS/MS tolerance: 20 ppm; missed cleavage: 2; fixed modification: carbamidomethyl; variable modification: oxidation; and database pattern: reverse. The cutoff of the global false discovery rate (FDR) for peptide and protein identification was set to 0.01. The transcript sequences of C. chilonis genes encoding the secreted ovarian proteins were submitted to the National Center for Biotechnology Information (http://www. ncbi.nlm.nih.gov/) and deposited under the accession numbers MH365478−MH366300. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository28 with the data set identifier PXD012914.

EXPERIMENTAL SECTION

Transcriptomic Analysis of Ovaries

The details of insect rearing are presented in Supplementary Methods. Transcripts originated from ovaries of female wasps at the later pupal (4 or 5 days after pupation, FPOvary) and adult (1 or 2 days after eclosion, FAOvary) stage, each with three biological replicates. RNA sequencing was performed by Novogene Bioinformatics Institute (Beijing, China) according to a previous study.10 Detailed procedures are presented in Supplementary Methods. Proteomic Analysis of the Secreted Ovarian Proteins

Ovaries of female adult wasps, aged 2 days, were carefully dissected and squeezed to release the fluids from the lateral oviducts in Pringle’s phosphate-buffered saline (PBS, pH = 7.4) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma, St. Louis, MO, USA). After centrifugation at 8000g for 10 min at 4 °C, the supernatant was filtered through a 0.22 μm Millipore filter. The filtrate was quantified with the BCA Protein Assay kit (Bio-Rad, USA) and then was subsequently stored at −80 °C until use. The proteomic analysis was performed by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China). The filter-aided sample preparation method was used,26 detailed procedures for which are presented in Supplementary Methods. The resulting peptides as a filtrate were desalted for each sample and loaded on C18 Cartridges (Empore SPE Cartridges C18 (standard density), bed I.D. Seven mm, volume 3 mL, Sigma). The peptides were concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g/L) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins. Each fraction was analyzed on a nano LC−MS/MS (liquid chromatography−mass spectrometry/mass spectrometry) system. Three μg peptide mixture was loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) connected to a C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The linear gradient was as follows: 0−55% buffer B for 110 min, 55−100% buffer B for 5 min, followed by a hold in 100% buffer B for 5 min. LC−MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to an Easy nLC instrument (Proxeon Biosystems, now Thermo Fisher Scientific) for 120 min. The mass spectrometer was operated in

Expression and Purification of Recombinant Proteins

Single-strand cDNA was synthesized from 1 μg RNA and used as template for polymerase chain reaction (PCR). The 5′ ends of cDNA sequences of C. chilonis IEP-1 and IEP-2B were identified by rapid amplification of cDNA ends (RACE) using a 5′ RACE kit (Takara-Clontech, CA, USA). At least one gene specific primer was designed for each partial IEP sequence (Table S1). All amplified PCR products were cloned into a pGEM-T Easy Vector (Promega Biotech Co., Ltd. Beijing, China) and sequenced. Recombinant Crp32B (rCrp32B) and C. suppressalis β-tubulin (rCs-β-tubulin) were both expressed in Escherichia coli. The softwares and Web sites used for sequence analysis and detailed procedures for protein expression, purification and dialysis are presented in Supplementary Methods. The purified rCrp32B was submitted to GenScript Co., Ltd. (Nanjing, China) as an antigen for immunization in rabbits. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot Analysis

Protein samples were subjected to the PAGE or polyacrylamide gradient gel electrophoresis (GenScript Co., Ltd., Nanjing, China) and stained with Coomassie Brilliant Blue R-250. The proteins separated by electrophoresis were transferred to a polyvinylidene difluoride membrane (Sigma, St. Louis, MO, USA). For Crp32B detection, we used rabbit anti-rCrp32B as the primary antibody (diluted 1:5000) and goat antirabbit IgG (H+L) (HuaAn Biotechnology, Hangzhou, China) as the secondary antibody (diluted 1:5000). For β-tubulin detection, we used a β-tubulin monoclonal antibody (Invitrogen, Carlsbad, CA, USA) as the primary antibody (diluted 1:5000) and goat antimouse IgG (H+L) (GenScript Co., Ltd., Nanjing, China) or 6× His-Tag HRP (HuaAn Biotechnology, Hangzhou, China) as the secondary antibody (diluted 1:5000). The immunoblot signal was detected by tetramethylbenzidine stabilized substrate for horseradish peroxidase (Promega, Madison, WI, USA). 2696

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Journal of Proteome Research Table 1. Proteins Involved in Cotesia chilonis Passive Avoidance isoelectric point/ molecular weight (kDa)

sequence length (signal peptide)

no. of peptides

no. of unique peptides

IEP-1/Cluster-8110.25416

7.51/34.72

325 (Y)

3

3

IEP-2A/Cluster8110.29162Comp39249_c0a IEP-2B/Comp42303_c0a

7.67/30.52

298 (Y)

9

9

7.94/34.47

324 (Y)

Crp32A/Cluster-8110.21682

8.24/26.70

242 (N)

8

8

Crp32B/Cluster-8110.27662

8.24/27.60

251 (N)

25

25

Crp32C/Cluster-8110.14141

8.98/27.08

246 (N)

12

12

protein name/gene ID

BLAST information (E-Value; GenBank No.) immunoevasive protein-1 [C. kariyai] (6 × 10−33; BAB72014.1) immunoevasive protein-2 [C. kariyai] (1 × 10−69; BAB72015.1) immunoevasive protein-2 [C. kariyai] (4 × 10−41; BAB72015.1) 32 kDa protein Crp32 [C. rubecula] (1 × 10−17; AAC31393.1) 32 kDa protein Crp32 [C. rubecula] (1 × 10−70; AAC31393.1) 32 kDa protein Crp32 [C. rubecula] (4 × 10−18; AAC31393.1)

accession no. MH365478 MH365479APD15629 MH365480APD15630 MH365481 MH365482 MH365483

a

Indicates a gene from reference.10 Y represents the deduced amino acid sequences with predicted signal peptides, N represents the deduced amino acid sequences without predicted signal peptides.

Quantitative Real-Time PCR (qPCR)

Japan). To compare the extent of encapsulation, capsules of beads were classified into six classes (0−5) as described by Teng et al. (2016).25 The encapsulation index (%) = [Σ(the number of beads with a defined encapsulated grade × its corresponding grade number)/(total number of beads observed × 5)] × 100.

Specific primers were designed using Primer3 Input (version 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/) (Table S1). The detailed protocol is presented in Supplementary Methods. 28S rRNA was used as a reference gene.10 The relative quantification in each tissue was calculated using the comparative 2−ΔΔCT method following the guidelines of Bustin et al.29,30 The results are presented as relative mRNA expression of three independent biological replicates.

Immunoprecipitation, Pull-Down Assay and LC−MS/MS

Immunoprecipitation and pull-down assay were performed using Pierce Classic Magnetic IP/Co-IP kit (Thermo Fisher Scientific, Wilmington, DE, USA) and Pierce Pull-Down PolyHis Protein: Protein Interaction kit (Thermo Scientific, Rockford, Illinois, USA) according to the manufacturer’s protocols, respectively. Detailed procedures are presented in Supplementary Methods. After SDS-PAGE analysis, five protein bands were cut out from the gel and sent to be commercially analyzed by mass spectrometry (Sangon Biotech, Shanghai, China). Briefly, each gel slice was digested with trypsin and lyophilized. An online Nano-RPLC was used on the Eksigent nanoLC-Ultra 2D System (AB SCIEX, USA). The samples were loaded onto a C18 nanoLC trap column (100 μm × 3 cm, C18, 3 μm, 150 Å) and washed with Nano-RPLC Buffer A (0.1% formic acid, 2% acetonitrile) at 2 μL/min for 10 min. An elution gradient of 5−35% acetonitrile (0.1% formic acid) in 90 min gradient was used on an analytical ChromXP C18 column (75 μm × 15 cm, C18, 3 μm, 120 Å) with a spray tip. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCIEX, USA) and a pulled quartz tip as the emitter (New Objectives, USA). Data were acquired using an ion spray voltage of 2.5 kV, a curtain gas pressure of 30 PSI, a nebulizer gas pressure of 5 PSI, and an interface heater temperature of 150 °C. For informationdependent acquisition, survey scans were acquired in 250 ms, and as many as 35 product ion scans were collected if they exceeded a threshold of 150 counts per second (counts/s) with a 2+ to 5+ charge-state. The total cycle time was fixed to 2.5 s. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of the peak width (18 s), and the precursor was then refreshed off the exclusion list. The resulting MS and MS/MS spectra were searched against the C. suppressalis transcriptome33 which has been deposited at DDBJ/EMBL/GenBank under the accession number GAJS00000000 in the MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.). The proteins were successfully identified based on 95% or higher confidence interval of their scores. The following search parameters were used: up to two missed cleavages allowed, carbamidomethyl

RNA Fluorescence in Situ Hybridization (RNA-FISH) and Immunofluorescence

RNA-FISH, immunohistochemistry and immunocytochemistry procedures were performed essentially as described previously,31,32 with detailed procedures presented in Supplementary Methods. The primary antibody was a rabbit anti-rCrp32B polyclonal antibody or a β-tubulin monoclonal antibody. The secondary antibody used was an Alexa Fluor 594 conjugated goat antirabbit IgG (Proteintech, USA), or an Alexa Fluor 568 conjugated goat antimouse IgG (Thermo Fisher Scientific, USA). Actin filaments and nuclei were stained with 1 × Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, Wilmington, DE, USA) and 1 μg/mL 4′-6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) respectively. Paraffin embedding and sectioning of ovarian calyx were performed by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). In Vivo Encapsulation Assay

We injected cOmplete His-Tag purification resin beads (Roche, IN, USA) or newly laid eggs of C. chilonis into C. suppressalis larvae to observe in vivo encapsulation. The beads were incubated with PBS, anti-rCrp32B serum (1:500), rCrp32B (50, 100, and 200 ng/μL), and eGFP (200 ng/μL) expressed in the same bacterial expression system with rCrp32B at 4 °C for 2 h. To mask the rCrp32B-covered beads with antibodies, the beads were also incubated with anti-rCrp32B serum (1:500) at 4 °C for another 2 h. Newly laid C. chilonis eggs were dissected out of the hemocoel of parasitized host larvae and washed with PBS at least three times. To mask the Crp32B on the egg surface, we incubated the eggs with anti-rCrp32B serum (1:500) at 4 °C for 2 h. All beads and eggs were washed three times in sterilized PBS before being injected into the hosts. Approximately 30 beads or newly laid eggs were injected into a host recipient, with five recipient larvae used for one treatment. Samples were recollected from recipient larvae 4 h after injection and observed under a phase contrast microscope (Nikon eclipse TS-100, 2697

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Figure 1. Expression profiles of Cotesia chilonis Crp32B. (A) Crp32B mRNA expression profile in different tissues of C. chilonis female adults. (B) Developmental expression profile of Crp32B mRNA in C. chilonis ovaries. Total RNAs (1 μg each) of different tissues and ovaries from different C. chilonis stages were reversed transcribed to cDNAs and used as templates for qPCR analysis, with the C. chilonis 28S rRNA gene used as an internal control. Data are presented as the means ± standard deviation. The different letters are significantly different based on one-way analysis of variance (ANOVA) and Tukey’s test with differences considered significant at P < 0.05. (C) The Crp32B expression profile in C. chilonis adults. Total proteins (0.5 μg each) from male adult, female adult different tissues were analyzed by 12% SDS-PAGE. (D) Developmental expression profile of Crp32B protein in C. chilonis ovaries. Total ovarian proteins (0.5 μg each) from different C. chilonis stages were analyzed by 12% SDS-PAGE. Crp32B was detected by immunoblotting using an anti-rCrp32B rabbit polyclonal antibody. β-Actin was used as an internal control.

cysteine as fixed modification, acetyl (protein N-term), deamidated, dioxidation and oxidation as variable modifications, 30 ppm for precursor ion tolerance and 0.15 Da for fragment ion tolerance. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http:// proteomecentral.proteomexchange.org) via the iProX partner repository28 with the data set identifier PXD012937.

statistical calculations were performed by Data Processing System (DPS) package (Version 9.5).34

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RESULTS

Identification of Passive Avoidance-Related Proteins

For the six libraries, 49.1−58.2 million clean reads were generated (Table S2), with 3607 FAOvary genes upregulated and 3625 downregulated compared to FPOvary genes. KEGG pathway analysis revealed that upregulated FPOvary genes mapped significantly to the ribosome, spliceosome, and proteins processing, which are related to gene transcription and translation. Compared to the FPOvary sample, cytokinecytokine receptor interaction genes were overrepresented in the FAOvary sample, with these genes being involved in innate immunity. We inferred that the adult wasps need more effective

Statistical Analysis

The differences between the means in qPCR analysis were compared using one-way analysis of variance (ANOVA) and Tukey’s test with differences considered significant at P < 0.05. The mean results for different treatments in the encapsulation assay were analyzed by Student’s t-test with differences considered significant at P < 0.05, P < 0.01, or P < 0.001. All 2698

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Figure 2. RNA-FISH analysis of Crp32B transcripts. (A−C) An ovary and (J−L) immature egg from female pupa were incubated with DEPC-treated water as controls; (D−I) An ovary and (M−O) immature egg from female pupa were hybridized with Cy3-labeled Crp32B mRNA probes (red). (G− I) Detailed views of the ovarian calyx of (D−F). The nuclei were stained with DAPI (blue).

immune defenses, since they may be much more exposed to pathogens compared to pupal wasps (Table S3). We identified 817 proteins as secreted ovarian proteins (SOPs) matched the FPOvary or FAOvary by LC−MS/MS

(Table S4), with 346 genes (42.35%) annotated in GO. At level 2, SOP genes were classified into ten molecular functional categories (Table S5). Ovarian proteins containing conserved enzymatic domains constituted a large proportion of secreted 2699

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Figure 3. Immunohistochemical visualization of endogenous Crp32B in the ovaries and on the egg surfaces of Cotesia chilonis adults. (A−C) Immunolocalization of Crp32B in a mature ovary. C is the detailed view of the box in B. (D) Immunolocalization of Crp32B in the ovarian calyx and on egg surfaces. Immunolocalization of Crp32B on the surfaces of immature eggs (E, F) and newly laid eggs (G, H). A rabbit anti-rCrp32B polyclonal antibody was used as the primary antibody (B, C, D, F, H) and the preimmune rabbit serum was used as the control (A, E, G). Alexa Fluor 594conjugated goat antirabbit IgG (red) was used as the secondary antibody. The nuclei were stained with DAPI (blue).

ovarian components. From the SOPs, we identified five proteins that are likely involved in passive evasion: IEP-1, IEP-2A, Crp32A, Crp32B, and Crp32C (Table 1). In our previous study, we identified two passive avoidance-related proteins, IEP-2A and IEP-2B, which were specifically expressed in the ovary and venom gland, respectively.10 The full-length cDNA sequences of IEP-1 and IEP-2B were obtained by 5′ RACE, which showed 100% identity to the sequences obtained from the transcriptomic data. Each IEP homologue has a predicted signal peptide at N-terminus, and different numbers of epidermal growth factor-like domains were identified (Figure S1A). These three Crp32 homologues have

no predicted signal peptides, and only one motif at N-terminus with high identity values was detected (Figure S1B). Expression Profiles of Crp32B

qPCR results showed that IEP-1 and three Crp32 homologue mRNA were all expressed at significantly higher levels in the ovary than other female adult tissues (Figure 1A and Figure S2A−C). After pupation of C. chilonis larvae, the mRNA levels of IEP-2A, IEP-2B and three Crp32 homologues increased in the ovary starting at day 2, peaking at day 3 or 4 and then decreasing at day 5 and remained at a low level at day 2 posteclosion (Figure 1B and Figure S2E−H). The level of IEP-1 mRNA peaked at day 1 after eclosion (Figure S2D). 2700

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Figure 4. In vivo encapsulation analysis of resin beads (A) and mature Cotesia chilonis eggs (B). Data are presented as means ± standard deviation. Significant differences are indicated by Student’s t-test (***, P < 0.001; NS, no significantly different).

We expressed rCrp32B in E. coli and highly enriched it using His-Tag resin beads for the production of the rabbit polyclonal antibody (Figure S3B). Western blot results showed that Crp32B was present in the ovary but not in other tissues (Figure 1C), which is different from the qPCR-based expression profiles (Figure 1A). This result is likely due to the low expression level of Crp32B. Similarly, although Crp32B was transcribed at day 2 postpupation (Figure 1B), Crp32B protein was not detected at this time point. Crp32B accumulated during the following days and peaked at day 2 posteclosion (Figure 1D). RNA-FISH results suggested that Crp32B was transcribed throughout the ovary, especially in the ovarian calyx. Crp32B transcripts were also expressed in nurse cells, follicular cells and oocytes (Figure 2). In addition, immunofluorescent staining revealed that Crp32B protein was present in all parts of the ovary and on the egg surface (Figure 3).

and 3 with an ion score of >100 but absent in bands 4 and 5 are shown in Table 2. More than half of the components listed in Table 2 are proteins containing conserved enzymatic domains, with β-tubulin being the most abundant component among these proteins. β-Tubulin (a single protein component with a molecular weight of 55 kDa) of C. suppressalis could be detected using a commercially purchased β-tubulin monoclonal antibody (Figure S5). The molecular weight of rCs-β-tubulin was higher than 55 kDa for an unknown reason. However, it could be recognized by the β-tubulin monoclonal antibody (Figure S6). The pull-down assay showed that rCs-β-tubulin could capture the anti-rCrp32B antibody (Figure 5C), and rCrp32B could cross react with the βtubulin monoclonal antibody (Figure 5D). We predicted continuous (linear) epitopes of Crp32B and Cs-β-tubulin with a window length of 10 using ABCpred (Table S7), which resulted in the identification of two groups of similar continuous epitopes (Figure 5E).

Effect of Crp32B on the Host Encapsulation Response

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The rCrp32B protein significantly prevented the beads from being encapsulated by host hemocytes dose-dependently as compared to the control beads that lacked rCrp32B or were covered with GFP. When rCrp32B-covered resin beads were masked with an anti-rCrp32B antibody, the encapsulation was significantly increased (Figure 4A). Moreover, the encapsulation extent of mature eggs was significantly enhanced when they were preincubated with the anti-rCrp32B antibody (Figure 4B).

DISCUSSION Using high-throughput sequencing, molecular biological and physiological approaches, we identified 817 secreted ovarian proteins of C. chilonis and presented evidence that one of five avoidance-related proteins, Crp32B, played a role in the passive evasion of parasitoid eggs. These results are supported by those of our previous studies at the physiological level in which C. chilonis was shown to use a combination of passive and active strategies to escape from the host cellular immune response.25 In previous studies, the ovary connected to the venom apparatus was chosen as the comparative tissue, because it clearly performs a distinct function in wasp reproduction.35 Burke and Strand identified differentially upregulated transcripts in the venom gland, teratocyte, and larval transcriptomes compared to those observed in ovaries.35 However, in some parasitic wasp species, secreted ovarian proteins play important roles in regulating host immune responses.7,20,36 Therefore, in this study, we carefully dissected and squeezed C. chilonis ovaries to collect the fluids from the lateral oviducts instead of pooling the samples from the entire ovaries, eliminating the effects of egg/cellular proteins to the greatest extent possible. Genes that

Identification of Proteins Antigenically Related to Crp32B in C. suppressalis

Western blot and immunofluorescence results indicated that antibodies against rCrp32B could cross detect several components in different areas of C. suppressalis larva, and two protein components with molecular weights of 70 and 55 kDa were labeled in all C. suppressalis tissues tested (Figure 5A and Figure S4). Immunoprecipitation followed by proteomics analysis was performed to identify host proteins having antigenic similarity to Crp32B. The proteins that were solely present in the anti-rCrp32B serum group (band 1, 2, and 3) but absent in the IgG group (band 4 and 5) were identified as components antigenically related to Crp32B (Figure 5B and Table S6). The peptides identified by MS/MS in at least one of the bands 1, 2, 2701

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Journal of Proteome Research

hemocoel by an endoparasitoid. We speculate that ovarian enzymes may be involved in host regulation or ovary metabolism in the endoparasitoid, although the exact role of these enzymes remains unknown. This is the first report in which a combination of transcriptomic and proteomic approach was used to identify the secreted ovarian proteins of a parasitoid wasp. This data set may provide further opportunities to investigate novel features of the secreted ovarian proteins. Endoparasitoid wasps use a mimicry strategy in the passive evasion of host immunity. For example, CkIEP-1 protects parasitoid eggs from encapsulation by host Pseudaletia separata hemocytes but not from hemocytes of larval cutworms, Spodoptera litura, an incompatible host for C. kariyai.19 Plasma proteins that cross-reacted with IEP antiserum are present in P. separata, but not in S. litura. However, the specific components that parasitoids mimic remain unclear. In the current study, among the 36 proteins identified in immunoprecipitates from C. suppressalis, Cs-β-tubulin is the most abundant component and it is antigenically related to Crp32B. To identify the site of the protein exposed to the host immune system, we performed multiple alignments of the predicted continuous epitopes in Cs-β-tubulin and Crp32B, although the whole amino acid sequences of these two proteins showed low similarity. We identified two groups of similar continuous epitopes exhibiting the same or very similar five-continuous amino acid regions that are likely involved in the cross detection. In future work, these epitope peptides should be synthesized as immunogens to generate antibodies that could aid in determining whether the predicted continuous epitopes are recognized by the antibodies. However, most antigenic determinants of proteins are discontinuous, and without 3D structural data of Crp32 homologues, it is difficult to predict the discontinuous (conformational) epitopes.39 Besides β-tubulin, it is unclear whether the 35 other proteins identified in the immunoprecipitates could also share similar epitopes with Crp32B, and this possibility should be further studied in the future. Although the extent of encapsulation of mature eggs was enhanced significantly when they were preincubated with the anti-rCrp32B antibody, some eggs remained unencapsulated. In contrast, in other parasitoid/host systems, most of the wasp eggs were encapsulated when the related protein was blocked by its antibody20 or knocked down by RNA interference.21 Thus, it appeared that one passive-related protein was enough to protect the eggs from encapsulation in other systems. However, our results regarding egg encapsulation indicated that there were likely other functional components involved in protecting C. chilonis eggs from host immune responses. In addition to Crp32B, the functions of other known passive-related proteins and components on the egg surface remain to be studied.

Figure 5. Analysis of Chilo suppressalis proteins antigenically related to Crp32B. (A) Immunodetection of the proteins antigenically related to Crp32B in different tissues of C. suppressalis. Proteins (3 μg each) from the whole body or different tissues of C. suppressalis were analyzed on a 4−12% gradient gel. Rabbit anti-rCrp32B antibody was used as a primary antibody. (B) 4−12% gradient gel of immunoprecipitated proteins antigenically related to Crp32B in C. suppressalis. The five sections of the gel that were cut out are indicated by the red boxes. Tryptic peptides extracted from each section were excised and analyzed by LC−MS/MS. IgG was used as a control. (C) 8% SDS-PAGE showing the interactions of recombinant C. suppressalis β-tubulin (rCsβ-tubulin) (bait) with the anti-rCrp32B antibody (prey) by pull-down assay. Lane 1: input (anti-rCrp32B-antibody). Lane 2: nontreated gel control (minus bait, plus prey). Lane 3: immobilized bait control (plus polyhistidine-tagged eGFP, minus prey). Lane 4: polyhistidine-tagged eGFP: anti-rCrp32B antibody interaction. Lane 5: immobilized bait control (plus rCs-β-tubulin, minus prey). Lane 6: rCs-β-tubulin: antirCrp32B antibody interaction. (D) Western blot analysis showing rCrp32B (3 μg) that was detected with the β-tubulin monoclonal antibody. (E) Alignment of similar predicted continuous epitopes of Crp32B and C. suppressalis β-tubulin.

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CONCLUSIONS In this study, we described the comprehensive transcriptome and proteome profiling of secreted ovarian proteins from a parasitoid wasp and identified proteins involved in passive evasion against the host immune response as well as host antigens exhibiting molecular mimicry with one of the passive avoidance-related proteins. The bioinformatic, molecular, and physiological data in this study provided valuable clues for understanding the functions of secreted ovarian proteins in successful parasitism and the interaction between parasitoids and their hosts. These results will be useful for improving parasitoid efficacy for the future management of rice pests.

were significantly upregulated in the ovary of Nasonia vitripennis showed GO enrichment for terms related to cell division and transcription, characteristic of the reproductive functions of the ovary.37 However, our results indicated that proteins containing conserved enzymatic domains constitute a large proportion of secreted ovarian components in molecular functional categories of the GO analysis, similar to that observed in parasitoid venoms.10,38 Thus, the results in this study may more accurately reflect the true components of ovarian fluids injected into host 2702

DOI: 10.1021/acs.jproteome.8b00824 J. Proteome Res. 2019, 18, 2695−2705

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Journal of Proteome Research Table 2. Immunoprecipitated Chilo suppressalis Proteins Identified by Mass Spectrometrya no.

GenBank ID

molecular weight

isoelectric point

no. of peptides

no. of unique peptides

1 2 3 4 5 6 7 8 9

GAJS01059437 GAJS01063451 GAJS01070710 GAJS01025742 GAJS01011104 GAJS01022830 GAJS01021389 GAJS01024731 GAJS01022263

30404 8296 51461 18815 47085 53166 17589 10644 49123

4.78 7.74 5.41 8.62 8.2 6.22 9.19 6.44 6.55

43 (33) 12 (8) 25 (7) 14 (6) 20 (7) 15 (6) 5 (2) 9 (4) 14 (7)

9 (9) 5 (4) 16 (5) 3 (2) 12 (7) 12 (6) 4 (2) 6 (3) 10 (6)

10 11 12 13 14

GAJS01068019 GAJS01023271 GAJS01017921 GAJS01011382 GAJS01070161

11565 48235 48096 38708 20105

10 5.81 6.66 7.23 5.66

5 (3) 31 (16) 24 (8) 16 (9) 10 (5)

4 (3) 23 (15) 20 (8) 13 (8) 7 (5)

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

GAJS01024516 GAJS01019375 GAJS01013427 GAJS01000632 GAJS01011252 GAJS01070552 GAJS01017121 GAJS01020835 GAJS01070219 GAJS01011491 GAJS01070376 GAJS01070500 GAJS01070699 GAJS01000076 GAJS01070436 GAJS01005495 GAJS01021408 GAJS01064328 GAJS01024993 GAJS01009609 GAJS01070621 GAJS01022971

46187 23092 10668 46540 39925 33437 20768 49556 22261 34122 26131 27524 48562 30886 28276 113672 29404 8715 44178 19700 36696 32333

5.31 7.15 6.75 8.39 5.34 7.74 4.47 8.68 5.12 8.66 6.01 5.43 8.81 8.93 8.74 9.13 8.77 5.3 8.16 6.28 4.94 6.84

20 (9) 13 (6) 11 (6) 13 (5) 10 (9) 18 (10) 5 (3) 21 (8) 14 (8) 7 (4) 9 (6) 13 (4) 11 (6) 10 (5) 10 (5) 8 (4) 11 (5) 11 (2) 16 (6) 4 (2) 7 (6) 5 (4)

11 (5) 8 (5) 7 (5) 6 (1) 8 (8) 10 (7) 4 (3) 15 (7) 9 (7) 6 (3) 5 (4) 11 (4) 8 (6) 9 (5) 8 (4) 6 (4) 7 (4) 5 (1) 11 (4) 2 (1) 4 (3) 5 (4)

description β-tubulin α-tubulin protein disulfide-isomerase tripeptidyl peptidase II tripeptidyl peptidase II signal recognition particle dihydrolipoamide dehydrogenase mitochondrial aldehyde dehydrogenase 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase short form ATP synthase regulatory particle triple-A ATPase 1 methionine-rich storage protein rho-associated protein kinase 1 strand H+ transporting ATP synthase beta subunit protease regulatory subunit 6A similar to AGAP001884-PA 6-phosphogluconate dehydrogenase similar to GA18629-PA tripeptidyl peptidase II heat shock protein 90 protein disulfide-isomerase A6 precursor Ribophorin eukaryotic translation initiation factor 4A AMP dependent CoA ligase V-type proton ATPase subunit H Enolase nucleolar protein 56 succinic semialdehyde dehydrogenase microtubule-associated protein futsch-like bifunctional aminoacyl-tRNA synthetase similar to CG6904-PA seminal fluid protein HACP059 phosphoglycerate kinase similar to ENSANGP00000015662 flocculation protein FLO11 isoform X1 dolichyl-diphosphooligosaccharide protein glycotransferase

band 1 score

band 2 score

band 3 score

661 161 153 134 128 119 116 103 102

1006 204 60 N 35 38 N 43 106

953 232 36 N 15 N N 123 62

101 N 26 N N

N 248 192 177 177

27 80 86 59 N

N N N N 54 33 N N N N N 31 N N 77 N N 40 N N 34 N

161 161 160 154 150 145 143 135 133 132 129 128 119 116 113 111 103 100 22 92 85 68

36 N 62 54 72 N N 22 72 N 54 137 30 N 120 55 21 84 135 129 105 105

a The peptides identified by MS/MS in at least one of the bands 1, 2, and 3 with an ion score of >100 but were absent in bands 4 and 5 are shown. N indicates that the protein was not identified in the corresponding band.

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Author Contributions

ASSOCIATED CONTENT

S Supporting Information *

Z.-W.T., Q.F., and G.-Y.Y. conceived and designed the experimental plan. Z.-W.T. and X.-H.Y. analyzed and interpreted the sequence and experimental data. Z.-W.T., H.Z.W., S.-J.X., G.X., and F.W. performed the experiments. Z.W.T. wrote the manuscript. Q.F. and G.-Y.Y. provided valuable suggestions regarding this work. All authors read and approved the final manuscript.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.8b00824. Supplementary Methods; Figures S1−S6 (PDF) Tables S1−S7 (XLSX)

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Notes

The authors declare no competing financial interest. The transcript sequences of C. chilonis genes encoding the secreted ovarian proteins were submitted to the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ ) and deposited under the accession numbers MH365478− MH366300. The mass spectrometry proteomics data in this paper have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+86)-571-88982696. Fax: (+86)-571-88982988. ORCID

Gongyin Ye: 0000-0003-4937-8867 2703

DOI: 10.1021/acs.jproteome.8b00824 J. Proteome Res. 2019, 18, 2695−2705

Article

Journal of Proteome Research iProX partner repository28 with the data set identifiers PXD012914 and PXD012937.

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ACKNOWLEDGMENTS Financial support was provided by National Natural Science Foundation of China (Grant No. 31801796, http://www.nsfc. gov.cn/), Major International (Regional) Joint Research Project of National Natural Science Foundation (Grant No. 31620103915, http://www.nsfc.gov.cn/), National Key R&D Program of China (No. 2017YFD0200400), China Postdoctoral Science Foundation (2018M632482), The Program for Chinese Innovation Team in Key Areas of Science and Technology of Ministry of Science and Technology of the People’s Republic of China (2016RA4008), Program for Chinese Outstanding Talents in Agricultural Scientific Research of Ministry of Agriculture and Rural Affairs of the People’s Republic of China. In addition, we would like to thank Prof. Fei Li (Institute of Insect Sciences, Zhejiang University) for kindly providing us the complete sequence of β-tubulin from the Chilo suppressalis genome.

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ABBREVIATIONS CkIEP, Cotesia kariyai IEP; Crp32, Cotesia rubecula protein 32; DAPI, 4′-6-diamidino-2-phenylindole; DEG, differentially expressed gene; DPS, Data Processing System; FDR, false discovery rate; FPOvary, transcripts originated from ovaries of later pupal female wasps (4 or 5 days after pupation); FAOvary, transcripts originated from ovaries of adult stage female wasps (1 or 2 days after eclosion); GO, Gene Ontology; IEP, immunoevasive protein; kDa, kiloDalton; LC−MS/MS, liquid chromatography−mass spectrometry/mass spectrometry; PBS, Pringle’s phosphate-buffered saline; PCR, polymerase chain reaction; PDV, polydnaviruses; pI, isoelectric point; RACE, rapid amplification of cDNA ends; rCrp32B, recombinant Crp32B was expressed in E. coli; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOP, secreted ovarian proteins; VLPs, virus-like particles.

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