Insect Molecular Biology

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Insect Molecular Biology (2014) 23(2), 255–268

doi: 10.1111/imb.12078

Structure and expression of a cysteine proteinase gene from Spodoptera litura and its response to biocontrol fungus Nomuraea rileyi

H. Chen*†, Y. Yin*, E. Feng*, X. Xie* and Z. Wang* *Genetic Engineering Research Centre, College of Life Science, Chongqing University, Chongqing, China; and †Institute of Plant Physiology and Ecology, Chinese Academy of Sciences Key Laboratory of Insect Developmental and Evolutionary Biology, CAS, Shanghai, China Abstract Cysteine proteinases (Cyps) play vital roles in many biological processes, including physiological and pathological reactions. In the present study, we cloned a full cDNA of SlCyp, encoding a 344-aminoacid protein from Spodoptera litura. The putative amino acid sequence shared >75% identity with Cyps from other insects. A phylogenetic analysis revealed that SlCyp is closely related to other known lepidopteran Cyps. Real-time PCR and Western blotting analyses showed that SlCyp is induced by Nomuraea rileyi infection in all the tissues tested. The strongest SlCyp mRNA and protein expression was found in haemocytes, followed by the fat bodies, of unchallenged and N. rileyi-challenged S. litura. A timecourse analysis showed that SlCyp mRNA and protein expression levels were upregulated in the haemocytes and fat bodies by N. rileyi infection. Upon N. rileyi infection, the proteolytic activities of SlCyp were also significantly higher in the haemolymph than in normal or phosphate-buffered-saline-challenged controls. These results suggest that SlCyp plays an important role in the innate immunity of S. litura in response to N. rileyi. SlCyp mRNA and protein expression and activities were also elevated during sixthinstar moulting and metamorphosis. Knocking down

First published online 28 January 2014. Correspondence: Prof. Zhongkang Wang, Genetic Engineering Research Center, College of Life Science, Chongqing University, 400030, China. Tel/fax: 86 023 65120489; e-mail: [email protected].

© 2014 The Royal Entomological Society

SlCyp transcripts with double-stranded RNA interference caused prepupal, pupal, and adult phenotypic changes, and SlCyp-silenced mutant larvae displayed a significantly lower survival rate after N. rileyi infection. These facts suggest that SlCyp plays a significant role in resisting N. rileyi infection and an essential role in larval development. Our data should facilitate the development of techniques for S. litura control. Keywords: cysteine proteinase, Nomuraea rileyi, Spodoptera litura, immunity.

Introduction Cysteine proteinases can be classified into different clans and families, depending on their molecular characters and substrate specificities. The cysteine proteinases of clan CA include papain and cathepsins L, B, S and H (Barrett & Rawlings, 2001; Cristofoletti et al., 2005). The cysteine proteinases of clan CD include legumains, caspases, separases, gingipains and clostripains (Leon et al., 2004). The different substrate specificities and protease inhibitors of clan CD allow them to be experimentally distinguished from clan CA (Leon et al., 2004). Cysteine proteinases are commonly found in animal lysosomes, and not only digest waste proteins but also participate in a number of life processes, including apoptosis during tissue differentiation (Isahara et al., 1999), and in abnormal physiological processes, including tumour transfer malignancy (Werle et al., 1999). Cysteine proteinases have been studied in various species of insects. In Coleoptera, Diptera, and Hemiptera, they are important digestive enzymes, and have been considered targets for pest control (Murdock et al., 1987; Matsumoto et al., 1995; Cristofoletti et al., 2003); however, a number of cysteine proteinases play key roles in developmental processes. For example, cathepsins B and L are thought to be important in embryogenesis (Uchida et al., 2001; 255

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Zhao et al., 2002, 2005) and tissue remoulding during insect metamorphosis (Hegedus et al., 2002; Liu et al., 2006; Wang et al., 2010). Cysteine proteinases are also reported to play an important role in host immune systems. The clan CA papain proteases and clan CD C13 legumain-like proteinases are involved in the major histocompatibility complex (MHC) class II antigen-processing pathway in mammals (Murray et al., 2005), and cathepsin L and legumain are involved in the adaptive immunity of vertebrates. Sarcophaga peregrine cathepsin L is present in haemocytes and participates in the degradation of foreign proteins during the immune response (Fujimoto et al., 1999). Spodoptera litura relies on the innate immune response only, but whether cathepsin L has a role in the innate immunity of Spodoptera is unknown. Spodoptera litura is an important polyphagous insect pest and is responsible for widespread economic damage to vegetables and ornamental plants in tropical and subtropical regions. This pest is difficult to control because it develops insecticide resistance, with consequent control failure (Kranthi et al., 2001, 2002; Ahmad et al., 2007). The fungus Nomuraea rileyi is the most important suppressor of S. litura in the field, and shows promise as a control for this pest. To better control S. litura using N. rileyi, an understanding of the molecular interactions between S. litura and N. rileyi is essential. When using subtractive hybridization to screen the immune-response-related genes in S. litura directed against N. rileyi, a cysteine proteinase gene (SlCyp) was isolated (Chen et al., 2012); however, no reports of SlCyp in S. litura have been published to date. In the present study, we attempted to identify the functions of SlCyp in S. litura by comparing its expression in normal and N. rileyi-infected S. litura.

Results Molecular cloning, characterization and structural analysis of the SlCyp gene Based on the expressed sequence tag determined with a suppression subtractive hybridization cDNA library, the full-length cDNA sequence of SlCyp (GenBank accession number: KC896759) was acquired by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE)-PCR, which generated an 1529-bp product that included a 150-bp 5′ untranslated region (UTR) and a 344-bp 3′ UTR (Fig. 1A). Sequence analysis showed that the full-length cDNA of SlCyp contained a 1035-bp open reading frame (ORF) encoding a protein of 344 amino acids (aa) that includes a 16-aa signal peptide. Analysis with EXPASY Server (http://www.expasy.org/) software showed that the protein’s isoelectric point was 6.67 and its molecular mass was 38.312 kDa.

A similarity search performed with BLAST revealed that the deduced aa sequence of the cDNA is homologous to cathepsin L. A cathepsin propeptide-inhibitor domain_I29 was identified at aa 27–87, which is located at the N-termini of certain C1 peptidases, including cathepsin L, where it acts as a propeptide. A Pept_C1 domain (cysteine proteinase clan CA, papain family C1 subfamily) was identified at aa 127–343, indicating that this protein is a cathepsin L of the papain family in the C1 subfamily of cysteine proteinases (Fig. 1A). The mature enzyme contains the catalytic triad C151–H290–N311, a feature conserved in all known cysteine proteinases (Berti & Storer, 1995). A multiple sequence alignment with the deduced protein sequence of SlCyp indicated that the SlCyp sequence has 99.4, 97.1, 89.2 and 75.5% similarity to Cyps from Spodoptera frugiperda (ADN19567.1), Spodoptera exigua (ABK90824.1), Helicoverpa armigera (AAQ75437.1) and Bombyx mori (AAB33990.1), respectively (Fig. 1B). Moreover, as shown in Fig. 1B, two putative N-glycosylation sites (N95 and N282) occur in the proregion and the mature enzyme, respectively. Two characteristic cathepsin L proteinase motifs are present in the proregion, E43–R47–Y51– N54–I58–N62 and K75–N77–Y79–D81 (Karrer et al., 1993). A G190–C191–N192–G193–G194 cluster, which is thought to be an important structural motif (Karrer et al., 1993), and an S340–P342–V344 motif, which may be important for cathepsin L secretion (Chauhan et al., 1998), were also found in the mature enzyme (Fig. 1B). To determine the relative position of SlCyp in evolution, a phylogenetic analysis of the Cyps from various species was performed with the neighbour-joining method using MEGA 5.0. The phylogenetic relationship analysis showed that the SlCyp protein is genetically closest to other available lepidopteran Cyps from S. frugiperda, S. exigua, and H. armigera (Fig. 1C). Consistent with this phylogeny, these proteins share high sequence identity (99.4, 97.1 and 89.2%, respectively). PCR was performed to isolate the genomic SlCyp sequence to investigate its structure. The full-length SlCyp genomic sequence is ∼6411 bp long and corresponds to a 1529-bp cDNA sequence. The exon/intron composition of the gene was determined by comparing the genomic sequence with the SlCyp cDNA sequence. The SlCyp gene comprises seven exons and six introns, which have a high AT content and typical 5′-GT splice donors and 3′-AG splice acceptors (Fig. 1D).

Expression and purification of recombinant SlCyp protein and preparation of antibodies To assess the SlCyp gene, the ORF of SlCyp (without the signal peptide) was cloned into the pET-30a(+) vector and expressed as a His-tagged fusion protein in Escherichia coli BL21(DE3) with isopropyl-1-thio-β-galactopyranoside © 2014 The Royal Entomological Society, 23, 255–268

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Figure 1. Characterization of the SlCyp gene. (A) Nucleotide and deduced amino acid sequences of Spodoptera litura cathepsin L. The predicted signal peptide cleavage site is indicated with an arrow. The C1 domain is underscored and the catalytic site (C151–H290–N311) is boxed. The cathepsin propeptide inhibitor domain I29 is doubly underscored. Two possible polyadenylation signals (TATAAA, ATTAAA) are shown in italics. (B) Comparison of the deduced amino acid sequences of SlCyp from S. litura, HaCyp (AAQ75437.1) from Helicoverpa armigera, BmCyp (AAB33990.1) from Bombyx mori, SfCyp (ADN19567.1) from Spodoptera frugiperda, and SeCyp (ABK90824.1) from Spodoptera exigua. The possible N-linked glycosylation sites are boxed. Conserved motifs E-R-Y-N-I-N, K-N-Y-D, G-C-N-G-G, and S-P-V are marked with the corresponding letters. (C) A phylogenetic tree was generated with MEGA version 5.0 from the Cyps of various species, including S. litura, S. frugiperda (ADN19567.1), S. exigua (ABK90824.1), H. armigera (AAQ75437.1), B. mori (AAB33990.1), Dermestes frischii (ABR88030.1), Aedes aegypti (XP_001655999.1), Delia coarctata (ACR56863.1), Tenebrio molitor (AAO48766.2), Drosophila melanogaster (NP_725347.1), Papilio polytes (BAM18960.1), Papilio xuthus (BAM17937.1), Macrobrachium nipponense (AEC22811.1), Sitophilus zeamais (BAA24444.1), Penaeus monodon (ABQ10739.1), Artemia salina (ABS17682.1), Artemia franciscana (AAV63977.1), Camponotus floridanus (EFN65237.1), Triatoma infestans (AAR12010.1), Litopenaeus vannamei (CAA68066.1), Rhodnius prolixus (AAL34984.1), Apostichopus japonicus (ABW98676.1) and Triatoma brasiliensis (AEM76722.1). (D) Lengths of the exons and introns in the genomic DNA from S. litura are indicated. Light grey and black highlight the exons and introns, respectively. UTR, untranslated region; ORF, open reading frame.

(IPTG) induction. Sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis (PAGE) analysis showed that the recombinant protein had a molecular mass of ∼47 kDa, which corresponds to the molecular mass calculated from the ORF (without the signal peptide) plus one S tag and two His tags encoded on the vector (Fig. 2). The soluble recombinant protein was further purified on Ni–NTA spin columns. The purified protein was characterized by Western blotting with an anti-His-tag antibody, and then injected into rabbits to generate specific antiserum. The total proteins from S. litura were used in Western blotting analysis to determine the specificity of the antibodies. The polyclonal antibody recognized both proSlCyp (38 kDa) and mature SlCyp (24 kDa) (Fig. 2), © 2014 The Royal Entomological Society, 23, 255–268

calculated according to the propeptide cleavage site at aa 126; therefore, the antiserum was suitable for further research. Tissue distribution of SlCyp The expression profiles of the SlCyp gene in different tissues were investigated at the transcription and translation levels with quantitative PCR (qPCR) and Western blotting, respectively (Fig. 3). The qPCR analysis showed that SlCyp was constitutively expressed in all the normal tissues examined. The highest expression was detected in haemocytes, then in the fat bodies, whereas the lowest expression was in the cuticle (Fig. 3A). After infection with

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H. Chen et al. 1–2 post inoculation are the key stages in fungal pathogenesis, when the hyphal bodies use diverse mechanisms to cope with the insect immune responses, and a large number of hyphal bodies were phagocytosed and encapsulated by haemocytes during this period, so that few hyphal bodies circulated freely in the haemolymph (Fig. 4B and C). By 3 days post inoculation, the hyphal bodies floating freely in the haemolymph had increased in concentration, accompanied by a reduction in the haemocyte counts (Fig. 4D).

Figure 2. Expression and purification of recombinant SlCyp fusion protein. Protein samples were analyzed with SDS-PAGE and stained with Coomassie Brilliant Blue. 1: Protein marker (kDa); 2: Extract-induced Escherichia coli BL21(DE3) containing empty PET-30a(+) as the control; 3: crude extract from isopropyl-1-thio-β-galactopyranoside-induced cells containing PET-SlCyp; 4: purified SlCyp fusion protein; 5: purified recombinant SlCyp characterized by Western blotting; 6: total protein from Spodoptera litura; and 7: Western blotting analysis of SlCyp from S. litura. Arrow 1 indicates the precursor of the enzyme and arrow 2 indicates the mature enzyme.

N. rileyi for 24 h, the expression of SlCyp was significantly induced in all the tissues tested compared with the normal control tissues (Fig. 3B); however, the translational expression profile differed slightly from the transcriptional profile. SlCyp protein was present in the haemocytes and fat bodies of normal S. litura. When N. rileyi was injected into the body cavities of S. litura larvae, SlCyp protein was detected in all the tissues tested. The highest expression levels were observed in haemocytes, then in the fat bodies (Fig. 3C), indicating that haemocytes and fat bodies are two major organs involved in the defence responses against N. rileyi. Interestingly, mature SlCyp and the SlCyp proenzyme showed different mobility, probably because the protein was post-translationally modified, a common characteristic of the cathepsin-L-like proteases of other insects, including H. armigera (Liu et al., 2006), Sarcophaga peregrine (Homma et al., 1994), Delia radicum (Hegedus et al., 2002), B. mori (Yamamoto et al., 1994) and Drosophila melanogaster (Tryselius & Hultmark, 1997).

Characterization of host haemolymph during N. rileyi infection and expression profiles of SlCyp during different infection stages As shown in Fig. 4B, N. rileyi had entered and colonized the host haemolymph 24 h after inoculation. Hyphal bodies (short hyphal lengths and yeast-like blastospores) had attached to the haemocytes and appeared to be stimulating haemocyte aggregation, and the hyphal bodies were being phagocytosed or encapsulated. Days

Figure 3. Tissue distribution of SlCyp in Spodoptera litura. (A) and (B) qPCR analysis of SlCyp expression patterns in different tissues from normal and Nomuraea rileyi-challenged S. litura 24 h post inoculation. Relative gene expression was normalized against the expression of two internal reference genes in each tissue sample. (C) Western blotting analysis of SlCyp distribution in different tissues from normal (upper panel) and N. rileyi-challenged S. litura 24 h post inoculation (lower panel). Values shown are the means ± SE of three experiments. * and ** indicate statistical significance at P < 0.05 and P < 0.01, respectively.

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Figure 4. Different patterns of haemocytes and hyphal bodies (HB) after inoculation with Nomuraea rileyi. (A) Normal haemocytes. (B) and (C) At 24 h and 48 h post inoculation, the presence of the fungus appeared to stimulate hemocyte aggregation. HB were phagocytosed and encapsulated by haemocytes. (D) At 72 h post inoculation, HB were floating free in the haemolymph.

To examine the expression profiles of the SlCyp gene activated by N. rileyi during different infection stages, we performed a time-course analysis of the larval haemocytes and fat bodies with quantitative real-time PCR (qPCR). The results showed that SlCyp expression increased in haemocytes and peaked 3 h after N. rileyi infection. The SlCyp transcript levels remained high until 48 h (Fig. 5A). The transcript levels of this gene were also upregulated in the fat bodies of S. litura 6 h after infection with N. rileyi and remained high at 12, 24 and 48 h (Fig. 5B); however, the transcript levels also increased in these two tissues in the phosphate-buffered saline (PBS)challenged controls, possibly in response to hydrostatic pressure and wounding. The expression patterns of the SlCyp protein were also examined in the larval haemocytes and fat bodies using Western blotting. The protein expression in the haemocytes of PBS-challenged controls remained at a normal level from 0 h to 12 h post © 2014 The Royal Entomological Society, 23, 255–268

inoculation, and then declined; however, this expression pattern contrasted with that observed after N. rileyi treatment. As shown in Fig. 5C, the protein levels of SlCyp increased significantly in haemocytes and remained high from 3 h to 48 h after inoculation with N. rileyi. In the fat bodies, the expression of SlCyp protein increased gradually and peaked at 24 h after treatment with N. rileyi. The protein levels also increased in the PBS-challenged control fat bodies, but the protein and transcripts were increased significantly more by N. rileyi infection than by PBS challenge in the fat bodies. The SlCyp mRNA and protein levels in these two tissues were significantly induced by the injection of a conidial suspension compared with their induction by a PBS injection, confirming that this protein plays a significant role in resisting N. rileyi infection. A synthetic substrate of SlCyp, benzyloxycarbonyl-Lphenylalany-L-arginine-4-methylcoumaryl-7-amide (Z-Phe

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Figure 5. Expression profiles and proteolytic activity of SlCyp in Spodoptera litura during different infection stages. qPCR (A and B) and Western blotting analysis (C) of SlCyp expression pattern in the haemocytes and fat bodies of S. litura induced with Nomuraea rileyi or phosphate-buffered saline (PBS). Total RNA and protein from S. litura injected with N. rileyi or PBS were extracted from haemocytes and fat bodies at different time points. (D) S. litura haemolymph proteolytic activity assay. Haemolymph from S. litura injected with N. rileyi or PBS was extracted at different time points, and was used to measure the total proteolytic activity against Z-Phe-Arg-MCA. Each value is the mean ± SE of three replicates. * and ** indicate statistical significance compared with the control (PBS injection) at P < 0.05 and P < 0.01, respectively.

-Arg-MCA), was used to examine the pattern of SlCyp proteolytic activity activated by N. rileyi in the haemolymph during different stages of infection. Its proteolytic activity was low at 0–3 h after treatment with N. rileyi, but increased sharply at 6 h post inoculation and remained high at 12–24 h post inoculation, followed by a decline at 48 h post inoculation (Fig. 5D). Its activity also increased in the PBSchallenged controls; however, the activity of this protein was significantly more upregulated by the injection of conidial suspension than by an injection of PBS.

SlCyp expression and proteolytic activity at different larval stages To examine the expression of the SlCyp gene at different developmental stages, qPCR was performed using RNA extracted from the first instar to the adult stage. Although SlCyp was expressed at all the stages examined, its

expression was dynamic during development. In the larval stage, the expression of SlCyp remained very low until day 3 of the sixth instar, but the mRNA levels increased to a peak on days 4–5 of the sixth instar, followed by a decline during the pupal stages, and an increase in the adult stages (Fig. 6A). The expression patterns of SlCyp were also examined at the whole-body level with Western blotting. The translational expression profile was similar to the transcriptional profile. No protein expression was detected during larval development until day 1 of the sixth instar. It increased gradually thereafter, and peaked on day 5 of the sixth instar. In the pupal and adult stages, the expression of SlCyp remained at a relatively high level (Fig. 6B). We used the specific substrate of cathepsin L, Z-PheArg-MCA, to examine the pattern of proteolytic activity in the whole body at different development stages, according to Liu et al. (2006). As shown in Fig. 6C, the activity of © 2014 The Royal Entomological Society, 23, 255–268

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Figure 6. Expression and proteolytic activity assay of SlCyp in whole bodies of Spodoptera litura larvae at different development stages. (A) Transcriptional levels of SlCyp. ACTB and GAPDH were used as the references genes. The total RNA was extracted from whole bodies of S. litura at each stage. (B) Western blotting analysis of the expression levels of SlCyp. (C) Proteolytic activity assay of SlCyp in whole bodies. Equal amounts of whole-body total proteins at each stage were separated for Western blot analysis and a proteolytic activity assay. Each value is the mean ± SE of three replicates.

SlCyp was low in the first-instar larvae and up to day 3 of the sixth instar larvae, but increased sharply on the last 2 days of the sixth instar, followed by a decline after pupation.

Effects of double-stranded RNA interference of SlCyp transcripts on resistance to N. rileyi To examine the function of SlCyp, double-stranded (ds)RNA directed against SlCyp was synthesized in vitro and injected into the hemocoels of sixth-instar larvae. RT-PCR and Western blotting indicated that the SlCyp dsRNA efficiently reduced the levels of SlCyp mRNA and protein (Fig. 7A) in the haemocytes and fat bodies. The haemolymph was collected 36 h post inoculation to examine the proteolytic activity of SlCyp, which decreased significantly in the haemolymph after the injection of SlCyp dsRNA compared with the injection of green © 2014 The Royal Entomological Society, 23, 255–268

fluorescent protein (GFP) dsRNA (Fig. 7B). Furthermore, SlCyp dsRNA resulted in abnormal and delayed development. All larvae injected with GFP dsRNA completed their metamorphosis from the sixth instar to pupae and their development into normal adults (Fig. 7C1, 2, and 3), but the larvae injected with SlCyp dsRNA were dead before pupation or formed abnormal pupae and adults (Fig. 7C). The dead larvae (23.3 ± 1.5%) showed no marked morphological abnormalities (Fig. 7C4). Some larvae (25.0 ± 2.5%) successfully entered the pupal stage, but the formation and development of the head and thorax were arrested. The abnormal pupae usually died within 72 h of abnormal pupation (Fig. 7C5). Some insects (11.7 ± 1.5%) moulted normally up to the pupal stage, but subsequently failed to complete the adult moult, resulting in deformed wings (Fig. 7C6). In contrast, larvae injected with the GFP dsRNA neither died nor formed abnormal pupae (Fig. 7C and D).

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Figure 7. Effects of SlCyp double-stranded RNA interference (dsRNAi) on the levels of transcripts from the SlCyp gene, and a survival assay of Spodoptera litura. (A) dsRNA (5 μg) was injected into each larva after ecdysis to the sixth-instar stage. Total RNA was extracted from the haemocytes and fat bodies of the treated larvae for transcript analysis by reverse transcription (RT)-PCR. In the RT-PCR analysis, ACTB amplified from the same RNA samples as the target gene was used as the internal control. The control samples were injected with GFP dsRNA. (B) Changes in proteolytic activity in the haemolymph of S. litura after treatment with SlCyp dsRNA. (C) Phenotypic changes in S. litura after treatment with SlCyp dsRNA. 1 and 4 are larval phenotypic changes; 2 and 5 are pupal phenotypic changes; 3 and 6 are adult phenotypic changes. (D) Bioassay of dsRNAi of SlCyp in S. litura. Following injection with SlCyp dsRNA, 20 larvae were inoculated with phosphate-buffered saline (PBS) containing 1 × 107 spores/ml N. rileyi conidia (DsSlCyp-infected); 20 larvae were treated with GFP dsRNA and PBS containing 1 × 107 spores/ml N. rileyi conidia (DsGFP-infected); 20 larvae were inoculated with PBS without N. rileyi conidia (PBS); 20 larvae were treated with diethylpyrocarbonate (DEPC) without dsRNA and PBS containing 1 × 107 spores/ml N. rileyi conidia (DEPC-infected); 20 larvae were inoculated with 1 × 107 spores/ml N. rileyi conidia only (infected); 20 larvae were treated with SlCyp dsRNA without fungal infection (dsSlCyp); and 20 larvae were treated with GFP dsRNA without fungal infection (dsGFP). The numbers of dead larvae were counted daily. (E) LT50 in the bioassay. Values shown are the means ± SE of three experiments. ** indicates statistical significance at P < 0.01.

© 2014 The Royal Entomological Society, 23, 255–268

SlCyp resists N. rileyi infection in S. litura The role of SlCyp in the resistance to fungal pathogens was investigated by measuring the survival rates after RNA interference (RNAi)-mediated silencing of SlCyp in sixth-instar S. litura larvae. The knockdown of SlCyp increased the mortality rate in the infected larvae (Fig. 7D). The time required to achieve 50% mortality (LT50) values of diethylpyrocarbonate (DEPC)-infected, GFP dsRNA-infected and fungus-infected S. litura were 5.4, 5.3, and 5.4 days, respectively, which were significantly longer than the LT50 of fungus-infected SlCyp-RNAi mutants (4.3 days, P < 0.01; Fig. 7E). These data suggest that SlCyp is required for the insect’s resistance to infection by N. rileyi. Discussion The strong pathogenicity of N. rileyi against S. litura has led to its use in insect pest management, but the mechanisms of the interactions between S. litura and N. rileyi remain unknown. In the present study, we described the stage specificity of the expression, localization and function of SlCyp in S. litura during N. rileyi infection. The SlCyp cDNA encodes 344 aa residues with >75% identity to the cathepsin L Cyp of other insect species, and a phylogenetic analysis suggested that SlCyp displays an obvious orthologous relationship with other lepidopteran cathepsin L Cyps (SfCyp, HaCyp, and SeCyp). One highly conserved Pept_C1 domain and a multiple sequence alignment demonstrated that SlCyp is a cathepsin L in the papain family of the C1 subfamily of Cyps. DNA and protein primary sequence analyses detected a consensus region shared with other insect cathepsin L Cyps at the active site triad C151–H290–N311 and the essential cathepsin L secretion motif S340–P342–V344 encoded at the carboxyl terminal of the protein (Chauhan et al., 1998). Instead of the classic polyadenylation signal (AATAAA), two variant signals (TATAAA and ATTAAA) are located in the 3′-UTR of the SlCyp cDNA, suggesting a selective expression pattern in this organism (Beaudoing et al., 2000). Like cathepsin L of H. armigera (Liu et al., 2006; Wang et al., 2010), the present data demonstrate a role for SlCyp in S. litura metamorphosis. First, both qPCR and Western blotting analyses demonstrated that SlCyp is selectively expressed in the fat bodies and haemocytes, tissues directly involved in pupation (Wang et al., 2010). Second, a strong correlation between larval SlCyp expression and pupation was demonstrated with qPCR and immunoblotting analyses. SlCyp expression was not detected after day 2 of the sixth instar, increased significantly before the pupation stages, and remained relatively high in the pupal and adult stages. These results indicate that SlCyp is strictly regulated during larval development. SlCyp activity changes in the whole body were fully synchronized with larval development, further suggesting that © 2014 The Royal Entomological Society, 23, 255–268

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SlCyp regulates larval development. Finally, the injection of SlCyp dsRNA into sixth-instar larvae resulted in abnormal metamorphosis. Cathepsin L was generally thought to be a type of digestive enzyme. There has been little research into its function in S. litura immunity until now. Cyps are reported to play an important role in the insect immune system. Among these Cyps, the most important are the clan CA papain proteases. In humans, the clan CA papain proteinases are reported to be involved in the MHC class II antigen-processing pathway (Murray et al., 2005). A cathepsin L in shrimp is also reported to be responsive to viral challenge (Robalino et al., 2007; Zhao et al., 2007; Ren et al., 2010). In the present study, the SlCyp transcript was strongly induced in all the tissues tested in N. rileyiinfected S. litura, but the strongest induction was in the haemocytes, followed by the fat bodies. The upregulation of SlCyp indicates its role in protecting the host against microbial infection. Haemocytes and fat bodies have been shown to play an important role in limiting infection in invertebrates (Plows et al., 2006; Chang et al., 2007; Hong et al., 2007; Chen et al., 2012), and are always selected as the tissues in which to investigate the fluctuations of immune-related genes. In the present study, haemocytes and fat bodies were used as the target tissues to examine the expression profiles of SlCyp after N. rileyi infection. A time-course analysis showed that the expression of SlCyp was significantly upregulated in the haemocytes and fat bodies. The activity of SlCyp was also significantly upregulated by injection with a conidial suspension. Furthermore, interruption of the expression of SlCyp transcripts in larvae that had just moulted to the sixth-instar stage resulted in increased sensitivity to N. rileyi and reduced LT50. The induction of SlCyp expression by N. rileyi infection supports its involvement in the host protection against microbial infection. Tests involving the inhibition of enzyme activity or RNAi showed that Cyps, especially cathepsin L, are required for successful moulting in insects. Cyps from entomopathogenic fungi, viruses and plants are also involved in degrading the cuticle or its analogous tissue, the peritrophic matrix, in lepidopteran insects, including Manduca. sexta, Trichoplusia ni, B. mori, S. frugiperda, and H. armigera (Ohkawa et al., 1994; Samuels & Paterson, 1995; Hawtin et al., 1997., Pechan et al., 2002., Liu et al., 2006., Wang et al., 2010). For example, cysteine proteinase Pr4, which can effectively hydrolyse the M. sexta cuticle, was first identified in Metarhizium anisopliae culture filtrates (Samuels & Paterson, 1995). Nucleopolyhedrovirus Autographa californica liquefies its lepidopteran insect host by secreting enzymes that degrade its cuticle, whereas insects infected with the same virus that lacked cathepsin L expression remained intact several days after death (Hawtin et al., 1997). The

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data obtained in the present study demonstrate that SlCyp plays a significant role in resisting N. rileyi infection and an essential role in larval metamorphosis. These observations could represent the basis of a novel strategy for S. litura control based on the rational design of chemicals and microbial pesticides targeting SlCyp to improve the efficacy of microbial pesticides against lepidopteran pests.

Experimental procedures Spodoptera litura and N. rileyi Spodoptera litura larvae were reared on an artificial diet at 27 ± 1 °C in 75 ± 5% humidity, with a photoperiod of 16 h light:8 h dark. Larvae and pupae were collected daily. N. rileyi strain CQNr129 was isolated from infected S. litura. The fungus was cultured on 1⁄4-strength Sabouraud dextrose agar medium (1/4 SDAY: 1% dextrose, 0.25% mycological peptone, 0.5% yeast extract, and 2% agar) for 10 days at 28 °C to produce conidia. A conidial suspension was prepared in PBS at 1 × 107 spores/ml.

Treatments and tissue collection Last-instar larvae were divided into two groups and anaesthetized on ice for 5 min before injection. Group 1 was challenged with 5 μl of a conidial suspension containing 1 × 107 spores/ml at the intersegment behind the second abdominal segment (Chen et al., 2012; Wang et al., 2012). Group 2 was challenged with 5 μl of PBS as the control. All the larvae were placed on ice at the indicated time points and carefully dissected to isolate their different tissues, such as the midgut, fat body, haemocytes, head and cuticle. These were then washed in DEPC-treated PBS solution for RNA and protein extraction, or immediately placed into liquid nitrogen and stored at −80 °C until use. To study the encroach process of spores in the hemocoele after N. rileyi inoculation, 50 μl of haemolymph was collected from the larvae at 1, 2, and 3 day post inoculation or from the normal control group. Slides were prepared for observation under an optical digital microscope.

RNA extraction, cDNA synthesis, and DNA preparation TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA from the pooled tissues of each larval group, according to the manufacturer’s protocol. DNAse-I-treated total RNA was converted to cDNA with the First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania), according to the manufacturer’s instructions. Genomic DNA was isolated from larvae using a Genomic DNA Purification Kit (Omega, Norcross, GA, USA), according to the manufacturer’s instructions.

Cloning the full-length cDNA of SlCyp and its application to genomic sequencing Based on the partial sequence of SlCyp (Chen et al., 2012), its 3′ and 5′ ends were obtained with the switching mechanism at the 5′ end of the RNA transcript (SMART)-RACE approach. 3′-RACE PCR was performed with a cDNA template from S. litura RNA using the gene-specific primers FCypf1 and CDSIII. For 5′-RACE PCR, the gene-specific primer FCypr1 and a 5′ PCR primer were

used. Both the 5′-RACE and 3′-RACE products were cloned into the pMD19-T Simple vector (TaKaRa Bio Inc., Shiga, Japan) and sequenced. Full-length SlCyp was obtained by overlapping the two fragments. To confirm the cDNA sequence assembled from the overlapping PCR products, the entire coding regions of SlCyp was amplified by PCR with the forward and reverse primers (FCypf and FCypr, respectively). To acquire the genomic DNA sequence of SlCyp, three pairs of primers (JCypf1/JCypr1, JCypf2/JCypr2, and JCypf3/JCypr3) were designed and synthesized according to the full-length cDNA of SlCyp. These PCR products were cloned and sequenced in the manner described above. The sequences of the PCR primer pairs, the expected sizes of the PCR products, and the cycling parameters for each PCR are listed in Table 1.

Bioinformatic analysis of SlCyp The conserved domains of SlCyp were detected with the help of bioinformatic tools available at the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/ Blast.cgi). We identified the ORF and performed a multiple alignment of homologues using the DNAMAN software 5.2.2. To reconstruct a phylogenetic tree of the Cyp proteins, the protein sequences of the candidate genes were used as query sequences to search for the homologous sequences of other species in the NCBI database (http://www.ncbi.nlm.nih.gov/). The phylogenetic tree of the predicted amino acid sequences of various Cyps of different species was constructed with the neighbor-joining method, implemented in MEGA 5.0, with a 1000 bootstrap replicates.

Recombinant expression and purification of SlCyp, and antibody preparation The ORF (without the signal peptide) of SlCyp was amplified with Pfu DNA polymerase (TaKaRa) using the primers BCypf and BCypr (Table 1), containing BamHI and HindIII restriction sites, respectively. The amplified fragments were cloned into the expression vector pET-30a(+) and then introduced into E. coli BL21(DE3) by transformation. A single positive colony was selected to be cultured overnight in Luria–Bertani (LB) medium with 50 μg/ml kanamycin at 37 °C. Then 1 ml of the E. coli culture was added to 100 ml of LB medium. When the optical density at 600 nm (OD600) reached 0.6, the culture was induced with 0.1 mM IPTG and cultured for a further 12 h at 17 °C. The cells were collected by centrifugation and then resuspended in 1 × binding buffer (50 mM sodium polyphosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0). The suspension was sonicated for 30 min on ice, and centrifuged again at 10 000 × g for 20 min at 4 °C. The suspension was subjected to SDS-PAGE to measure the expression of the target recombinant protein. The suspension was also purified under natural conditions with Ni2+–NTA binding resin (Qiagen, Shanghai, China), according to the manufacturer’s instructions, and SDS-PAGE was used to analyse the purified fusion protein. The concentration of the fusion protein was determined with a bicinchoninic acid (BCA) protein assay. To generate SlCyp polyclonal antibodies, 400 μg of purified SlCyp in 1 ml of PBS was mixed with 1 ml of Freund’s complete adjuvant and hypodermically injected into the backs of rabbits at two-week intervals. After the fourth immunization, the antibody titres were assessed with an enzyme-linked immunosorbent © 2014 The Royal Entomological Society, 23, 255–268

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Table 1. Primers used in this study

Primer pairs

Primer sequence

5′- and 3′-RACE PCR FCypf1 ACGAGGCAGTTGATGACAAG CDSIII ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N–1N FCypr1 CACCTTGTGGGATGTCTACG 5′ PCR AAGCAGTGGTATCAACGCAGAGT Cloning SlCyp fragments FCypf CAGCGTTGTAGTGGTAGGAC FCypr GTTGCTTTACTTAATTACTCTGC JCypf1 AATCGCAGCGTTGTAGTGG JCypr1 AACCGCTACGCCCTTCAT JCypf2 ATGAAGGGCGTAGCGGTT JCypr2 GTTGTCCATGAGGCCGCCGTTGC JCypf3 GCAACGGCGGCCTCATGGACAAC JCypr3 GATGCCGCAGTGGTTGTTCTTG Heterologous expression of SlCyp proteins BCypf TTAGGATCCGTCCGGGAGGAATGGAATGC BCypr ATAAAGCTTGAGTGGGTAGGAGGCGCTGGATGC SYBER-Green qRT-PCR DCypf CACTAGCACCTAGCAGTTCGAC DCypr AGACACGACAACTTCGTGGA Dactinf TGAGACCTTCAACTCCCCCG Dactinr GCGACCAGCCAAGTCCAGAC DGapdhf GTATGGCTTTCCGTGTTCCT DGapdhr TGACCTTCTGCTTGATAGCG DsRNA synthesis TCypf TAATACGACTCACTATAGGGAGTGCAGGTACAACCCCAAG TCypr TAATACGACTCACTATAGGGGTAGCCCACCACCATCACTC TGfpf TAATACGACTCACTATAGGGAGGGTGAAGGTGATGC TGfpr TAATACGACTCACTATAGGGCTTGAAGTTGGCTTTGAT First stand cDNA synthesis OligodG AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG CDSIII ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N–1N

PCR product size (bp)

Tm (°C)

Description

730

61

883

58

3′ RACE forward primer 3′ RACE universal adaptor primer 5′ RACE reverse primer Abridged universal amplification primer

1415

60

1147

59

3827

60

1088

61

963

62

Expression of protein forward primer Expression of protein reverse primer

125

60

178

60

99

60

quantitative RT- PCR forward primer Quantitative RT- PCR reverse primer Standard control forward primer Standard control reverse primer Standard control forward primer Standard control reverse primer

224

61

403

59

/ /

65 65

Full-length cDNA forward primer Full-length cDNA reverse primer Genomic sequence forward primer Genomic sequence reverse primer Genomic sequence forward primer Genomic sequence reverse primer Genomic sequence forward primer Genomic sequence reverse primer

SlCyp dsRNA synthesis forward primer SlCyp dsRNA synthesis reverse primer GFP dsRNA synthesis forward primer GFP dsRNA synthesis reverse primer First stand cDNA synthesis anchor primer First stand cDNA synthesis adaptor primer

RACE, rapid amplification of cDNA ends. Underline shows the HindIII (AAGCTT) and BamHI (GGATCC) restriction enzyme sites in the forward and reverse primer, respectively.

assay. The rabbit immunoglobulin G fraction was precipitated from the antiserum with 50% saturated ammonium sulfate and purified with DEAE–Sepharose column chromatography. The concentration of the resulting purified antibody in PBS was determined using an ultraviolet spectrophotometer, with sterile-filtered 0.02% sodium azide as the antiseptic.

Semiquantitative RT-PCR and fluorescence qPCR Total RNA was extracted from the samples after different treatments and treated with RNase-free DNase (Fermentas). DNAseI-treated total RNA (2 μg) from each sample was used to synthesize the first-strand cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas), following the manufacturer’s instructions. The resulting cDNA was used as the template for RT-PCR and qPCR with the SlCyp-specific primers DCypf and DCypr. The PCRs were performed at 94 °C for 30 s, followed by 25 cycles of amplification (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s) using 1 μl of first-strand cDNA in each 25 μl reaction mixture. Equal amounts of amplified products were analysed on 2% agarose gel. qPCR was performed using 1 μl of first-strand cDNA in each 25 μl reaction mixture. The cycling conditions using SYBR Green detection were 95 °C for 15 s followed by 40 cycles at 95 °C for 5 s, 58 °C for 15 s, and 72 °C for 30 s. After cycling was complete, a melting curve analysis was © 2014 The Royal Entomological Society, 23, 255–268

performed at 58 °C. Two reference genes, the β-actin gene (ACTB) and the glyceraldehyde phosphate dehydrogenase gene (GAPDH) (Guo et al., 2009; He et al., 2012; Meng et al., 2013) were used to normalize the target gene expression and correct for sample-to-sample variation. Amplification and expression quantification were performed using the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). Relative expression was calculated using the comparative cycle threshold method (Pfaffl, 2001), with normalization of the data to the geometric average of the reference genes (Vandesompele et al., 2002). A six-fold dilution series of pooled cDNA was used to assess the efficiency of the qPCR reaction for each gene-specific primer pair. A no-template control was also included to detect possible contamination. Experiments were performed using three biological and three technical replicates for each gene. The primers used for RT-PCR and qRT-PCR are shown in Table 1.

SDS-PAGE and Western blotting analysis Insect tissues and whole bodies were homogenized in insect physiological saline and centrifuged at 4 °C at 12 000 × g for 15 min. Supernatants containing soluble proteins were stored at −80 °C. Protein samples were denatured at 100 °C for 5 min, and then centrifuged at 10 000 × g for 15 min. The protein concentration was determined by BCA protein assay. SDS-PAGE was

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performed in 10–12% acrylamide gels in Tris-glycine–SDS buffer. The gel was stained with Coomassie Brilliant Blue R-250. For Western blotting, the proteins were transferred from the acrylamide gels to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and the blots were incubated with rabbit anti-SlCyp antibody, followed by incubation with a secondary goat horseradish-peroxidase-conjugated antibody. The bands were detected with the ECL Plus Western Blotting Detection Reagents (Pierce, Rockford, IL, USA).

Proteolytic activity assay The proteolytic activity was assayed according to Liu et al. (2006), using a specific substrate of cathepsin L, Z-Phe-Arg-MCA (Sigma, St. Louis, MO, USA). Protein extracts (10 μg) or haemolymph (2 μl) were preincubated at 37 °C for 10 min in 80 μl of sodium acetate buffer (pH 5.5), containing 340 mM sodium acetate, 60 mM acetic acid, 4 mM ethylenediaminetetraacetic acid, and 8 mM dithiothreitol to activate the cysteine proteases. They were then incubated for another 10 min after 10 mM Z-PheArg-MCA was added, then 200 μ1 of stop buffer (100 mM sodium acetate, 100 mM acetic acid, and 100 mM chloroacetic acid, pH 4.3) was added to terminate the reaction. The fluorescence of the liberated aminomethylcourmarin molecules was measured with a fluorometer at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. To inhibit the proteolytic activity, 10 μM of the cysteine protease inhibitor E-64 was used. The appropriate blanks and bovine serum albumin were processed simultaneously as negative controls. All the assays were repeated three times.

RNA interference and the survival assay The dsRNA was prepared as previously described (Wang et al., 2012). One pair of primers (TCypf and TCypr) was designed to synthesize the 224-bp region of the SlCyp gene that includes the T7 promoter region on both the sense and antisense strands. The quantity of SlCyp dsRNA was determined spectrophotometrically at 260 nm and by agarose-gel analysis. GFP dsRNA was generated with the same method and used as the negative control. The final dsRNA was dissolved in DEPC-treated water, stored at −80 °C, and used within 1 week. The primers used for dsRNA synthesis are listed in Table 1. Larvae that had just moulted to the sixth-instar stage were used for dsRNA injection. The larvae were anaesthetized on ice for 5 min before injection. dsRNA (5 μg) was injected into the larvae 6 h before inoculation with 1 × 107 spores/ml N. rileyi conidia, at the intersegment behind the second abdominal segment. Sixty larvae in each of three replicates were injected for each of the treatment groups. The larvae were then returned to their artificial diet and reared at 27 °C until they were killed for sample collection at the indicated time points. The samples were then used for RNA and protein analyses. The haemolymph, haemocytes and fat bodies were collected from 30 individual larvae for RNA, protein and proteolytic activity analyses. Twenty larvae were reared for the survival assay. We used another six injection controls: (1) DEPC-treated water with GFP dsRNA in the primary injections (dsGFP-infected); (2) DEPC-treated water without dsRNA in the primary injections (DEPC-infected); (3) PBS without N. rileyi conidial suspension in the secondary injections (PBS); (4) 20 larvae were treated with SlCyp dsRNA without fungal

infection (dsSlCyp); (5) 20 larvae were treated with GFP dsRNA without fungal infection (dsGFP); and (6) 20 larvae were inoculated with N. rileyi conidial suspensions only (infected). The larval mortality rate was recorded every day for 10 days, and the mean 50% lethal times (LT50) were estimated. All assays were performed three times. Statistical analysis The quantitative data are presented as means ± SE for each experiment. Student’s t test (SPSS version 17.0; SPSS, Chicago, IL, USA) was used to compare the relative expression of each gene between the controls and the challenged S. litura at each sampling point. The fold expression was analysed using one-way ANOVA to determine the significance of differences in each gene between the different exposure periods.

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© 2014 The Royal Entomological Society, 23, 255–268

Structure and expression of a cysteine proteinase gene from Spodoptera litura and its response to biocontrol fungus Nomuraea rileyi.

Cysteine proteinases (Cyps) play vital roles in many biological processes, including physiological and pathological reactions. In the present study, w...
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