A putative actin destabilizer, CpBV-RTX, encoded in Cotesia plutellae bracovirus andits expression in the parasitized Plutella xylostella

Hosanna H. Kim,Karen Barandoc,김용균 한국응용곤충학회 2008 Journal of Asia-Pacific Entomology Vol.11 No.2

A novel gene was identified in Cotesia plutellae bracovirus (CpBV), which acts as a symbiotic virus of the endoparasitoid wasp C. plutellae. The gene was encoded in the genome of CpBV, but not in the genome of its natural host, Plutella xylostella. The gene has an open reading frame comprised of 94 amino acids. A bioinformatic analysis indicated that the gene encodes a signal peptide comprised of 18 residues at its amino terminus and 4 glycosylation sites at threonine and serine residues, as well as two asparagine residues. The expression of the gene by C. plutellae was specific in parasitized P. xylostella, but not in unparasitized larvae. In parasitized larvae, the expression of the gene occurred on the first day after parasitization, and its expression resumed on the fourth day. The gene showed tissue specific expression in the fat body and epidermis, but not in hemocytes and gut tissue. Its sequence showed some similarity with that of a bacterial toxin, RTX, of Vibrio spp., especially in its actin cross-linking domain. The viral gene has been named CpBVRTX, and its putative physiological function is discussed in terms of host-parasite molecular interactions. A novel gene was identified in Cotesia plutellae bracovirus (CpBV), which acts as a symbiotic virus of the endoparasitoid wasp C. plutellae. The gene was encoded in the genome of CpBV, but not in the genome of its natural host, Plutella xylostella. The gene has an open reading frame comprised of 94 amino acids. A bioinformatic analysis indicated that the gene encodes a signal peptide comprised of 18 residues at its amino terminus and 4 glycosylation sites at threonine and serine residues, as well as two asparagine residues. The expression of the gene by C. plutellae was specific in parasitized P. xylostella, but not in unparasitized larvae. In parasitized larvae, the expression of the gene occurred on the first day after parasitization, and its expression resumed on the fourth day. The gene showed tissue specific expression in the fat body and epidermis, but not in hemocytes and gut tissue. Its sequence showed some similarity with that of a bacterial toxin, RTX, of Vibrio spp., especially in its actin cross-linking domain. The viral gene has been named CpBVRTX, and its putative physiological function is discussed in terms of host-parasite molecular interactions.

Cotesia plutellae Bracovirus Suppresses Expression of an Antimicrobial Peptide, Cecropin, in the Diamondback Moth, Plutella xylostella, Challenged by Bacteria

Barandoc, Karen P.,Kim, Jae-Hyun,Kim, Yong-Gyun 한국미생물학회 2010 The journal of microbiology Vol.48 No.1

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12-24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp's symbiotic virus.

Differential expression profile of genes encoded in a genome segment of Cotesia plutellae bracovirus in a parasitized host, Plutella xylostella

Wael GAD,최재영,제연호,김용균 한국곤충학회 2008 Entomological Research Vol.38 No.1

The Polydnaviruses are an insect DNA virus group. The segmented genome of a polydnavirus is located on the host wasp chromosome as a provirus. After replication, virions are delivered into the parasitized host, where their expression products play significant roles in specific processes of parasitism. However, little is known about how viral gene expression is controlled. In the present study, we tested whether different genes in a genome segment are expressed concomitantly. Cotesia plutellae bracovirus (CpBV) is a polydnavirus mutualistic to an endoparasitoid, Cotesia plutellae (Braconidae: Hymenoptera). Its circular genome segments in viral particles were captured by a transposon containing pUC origin sequence and replicated in Escherichia coli. CpBV-S30 (23.5 kb), one of the large CpBV genome segments, was fully sequenced, and seven open reading frames (ORF) were predicted. We analyzed expression patterns of the seven ORF in the diamondback moth, Plutella xylostella, parasitized by C. plutellae. Transcription analysesindicated that all ORF of CpBV-S30 in the parasitized P. xylostella were expressed from the first day of parasitization and then expression levels decreased over the period of parasitization, except for CpBV-H4, which showed an additional expression peak. In terms of tissue specificity in their expression patterns, all ORF were found to be expressed in the fat body, hemocytes and gut of the parasitized host. Promoter sequence analysis showed that all seven ORF had typical promoter elements, including TATA binding sites, but promoter component numbers and kinds varied. These results suggest that genes in a genome segment of CpBV can differentially express in the parasitized host, presumably by their different promoter components.

A Short Review of Teratocytes and Their Characters in Cotesia plutellae (Braconidae: Hymenoptera)

Basio Neil A.,Kim Yonggyun Korean Society of Applied Entomology 2005 Journal of Asia-Pacific Entomology Vol.8 No.2

An endoparasitoid wasp, Cotesia plutellae, oviposits into host hemocoe1 along with maternal factors including polydnavirus, ovarian proteins, and venom to manipulate host immune and development. This research reports presence and characters of teratocytes derived from developing embryos of C. plutellae. Teratocytes of C. plutellae could be cultured in ExCell-400 culture medium supplemented with antibiotics. Though they did not increase in numbers, they grew in size from $15.5\;{\pm}\;0.6\;{\mu}m$ during hatching to $3.72\;{\pm}\;9.6\;{\mu}m$ at 8 days after hatching in ExCell 400 medium. This report also introduces main characters about teratocytes found in other species.

Calreticulin in Cotesia plutellae Suppresses Immune Response of Plutella xylostella (L.)

Wook Hyun Cha,Yonggyun Kim,Dae-Weon Lee 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host

Gene Expression of Cotesia plutellae Bracovirus EP1-like Protein (CpBV-ELP1) in Parasitized Diamondback Moth, Plutellae xylostella

Lee, Kee-Woo,Cho, Sung-Hwan,Lee, Hyuk-Soo,Choi, Jae-Young,Je, Yeon-Ho,Kim, Yong-Gyun Korean Society of Applied Entomology 2005 Journal of Asia-Pacific Entomology Vol.8 No.3

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). This on-going research has identified several open reading frames (ORFs) including an EP1-like protein (ELP1). This study was intended to analyze gene expression of CpBV-ELP1 in the parasitized diamondback moth, Plutella xylostella. CpBV-ELP genomic DNA contains one intron (778 bp long). Its ORF consists of 726 bp encoding 241 amino acid residues. The hypothetical CpBV-ELP1 protein is predicted as 27,787.83 Da and possesses N-terminal signal peptide plus three potential N-glycosylation sites. Its amino acid sequence exhibits high homology with EP1 genes from C. congregata or C. karyai bracovirus. A reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that CpBV-ELP1 was expressed only in P. xylostella parasitized by C. plutellae. The expression levels were measured by real time quantitative RT-PCRs during entire parasitization period at $25^{\circ}C$ culturing temperature. The expression began at the first day of parasitization and increased with the parasitization period. The ORF PCR product was cloned, over-expressed, and molecular weight of the purified protein was about 30 kDa.

Identification of a Cys-motif Gene, TSP13, as a Putative Host Translation Inhibitory Factor in Plutella xylostella-Cotesia plutellae

Eunseong Kim,Yeongtae Kim,Yonggyun Kim 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.04

Translational control is a strategy for various viruses to manipulate their hosts to suppress any acute antiviral activity. Some cys-motif genes encoded in polydnaviruses or teratocytes act as host translation inhibitory factor (HTIF) to defend the host antiviral activity. A novel cys-motif gene, TSP13, was encoded in the genome of an endoparasitoid wasp, Cotesia plutellae. TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value at 7.928. Genomic DNA region encoding open reading frame is interrupted with three introns. TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected to nonparasitized P. xylostella. In the virus-injected P. xylostella, TSP13 was shown to be expressed by RT-PCR analysis. Thus, TSP13 was turned out to be encoded in the proviral CpBV genome. TSP13 was cloned into a eukaryotic expression vector, which was then used to infect Sf9 cells to transiently express TSP13. The synthesized TSP13 was detected in the culture broth. Purified TSP13 significantly inhibited cellular immune responses. Furthermore, TSP13 entered the target cells and was localized in the cytosol. This study reports a novel cys-motif gene, which is encoded in CpBV genome localized on chromosome(s) of C. plutellae and replicated to be encapsidated in the episomal viral particles during parasitization.

Cotesia plutellae Bracovirus Genome and Its Function in Altering Insect Physiology

Kim, Yong-Gyun,Choi, Jae-Young,Je, Yeon-Ho Korean Society of Applied Entomology 2007 Journal of Asia-Pacific Entomology Vol.10 No.3

Polydnavirus is a group of animal DNA virus mutually associated with some ichneumonoid wasp. Its relatively large size of genome has been considered as a major source of the parasitoid function to manipulate developmental and immunological processes of target parasitized insects. Cotesia plutellae bracovirus (CpBV) is a polydnavirus derived from C. plutellae, which parasitizes the diamondback moth, Plutella xylostella. Parasitized P. xylostella exhibits altered physiological symptoms in development and immune reactions. Though several other parasitic factors such as ovarian proteins, venom, and teratocytes are identified, CpBV has been more focused on elucidating various host physiological alterations occurring due to the parasitism, which has driven the CpBV genome project. CpBV attains a typical bracovirus structure by its single unit membrane envelope, in which multiple nucleocapsids are enclosed. Its genome DNAs are segmented and located on the genome of C. plutellae. Its replication begins at adult tissue development during pupal stage. An apparent genome size is 471 kb estimated from 27 segments separated on 5% agarose gel. A current work on the genome has been completely sequenced 24 genomic segments and analyzed their genomic structure. The aggregated genome size is 351,299 bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3% and 125 putative open reading frames are predicted. Though more than half (52.5%) of predicted genes are annotated as hypothetical, the annotated CpBV genes share amino acid sequence homologies with those of other bracoviral genomes. The annotated genes are classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode $I{\kappa}{\beta}-like$ and EP1-like, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Finally, two homologous genes, $CpBV15{\alpha}/{\beta}$, are unique in CpBV genome, which are not matched to any other known polydnaviral genes. Their homology with malarian circumsporozoite toxin and eukaryotic translation inhibition factors suggests their function in host translation inhibitory factor. This review discusses CpBV genes on their putative physiological functions based on the molecular interactions between the host-parasite.

Transient expression of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

Bokri Park,Yonggyun Kim 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp encoding two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28~50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. ORF301 and ORF302 were transiently expressed in hemocyte, fat body, gut, and epidermis of P. xylostella. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to non parasitized larvae of P. xylostella, in which the two genes were expressed at least for four days post-injection. The P. xylostella larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population, suppression of immune associated genes including cecropin, pro-phenoloxidase (PO) and serpin1, and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression by the viral segmenttreated larvae, but not by ORF302 specific dsRNA. The larval injected with CpBV-S3 exhibited an enhanced susceptibility to baculovirus infection. These results indicate that ORF301 of CpBV-S3, which containing BEN domain, suppresses both cellular and humoral immune responses in P. xylostella.

Attenuation of antiviral activity of the diamondback moth, Plutella xylostella, by a polydnaviral product, CpBV-IkB

Sungwoo Bae,Yonggyun Kim 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.05

The diamondback moth, Plutella xylostella, is reluctant to a baculovirus, Autographa california nucleopolyhedrosis virus (AcNPV) at its oral administration. However, parasitization by an endoparasitoid wasp, Cotesia plutellae, enhances the viral susceptibility. This study analyzed an antiviral activity of P. xylostella in response to the viral infection and determined the parasitic factor inhibiting the antiviral mechanism. For the analysis of antiviral activity of P. xylostella, a recombinant AcNPV expressing enhanced green fluorescence (AcNPV-EGFP) was orally adminstered to lavae of P. xylostella. After 24 h, EGFP expression was observed in the midgut tissue at a confocal-FITC mode. At the same time, a characteristic midgut melanotic response (MMR) was observed in some midgut regions under a phase contrast microscope. Thereafter, the EGFP signal was attenuated, while MMR spread on most midgut region. When the MMR was scored from 0 to 5 by the intensity of melanized cell density, it increased in time- and dose-dependent manners at the viral administration per os. These results suggest that the MMR is an antiviral activity of P. xylostella. This antiviral activity was significantly attenuated by C. plutellae parasitism. The parasitized P. xylostella showed significant decrease in the MMR score compared to nonparasitized larvae when they were orally administered with the same dose of AcNPV. To determine the parasitic factor(s) inhibiting the antiviral activity from the symbiotic polydnavirus of C. plutellae (C. plutellae bracovirus: CpBV), CpBV-IkB, which is a viral homolog of NFkB inhibitor and has been considered as an antiviral factor as in other polydnaviruses, was tested. A recombinant AcNPV expressing CpBV-IkB (AcNPV-IkB) was constructed and administered to P. xylostella larvae. As expected, AcNPV-IkB significantly decreased the antiviral activity measured by the MMR score compared to AcNPV-EGFP treatment. This study suggests that CpBV-IkB plays an antiviral parasitic role in the molecular interactions between P. xylostella and C. plutellae.