Rahul Hepat

RNA interference of a heat shock protein, Hsp70, loses its protection role in indirect chilling injury to the beet armyworm, Spodoptera exigua.

The beet armyworm, Spodoptera exigua, is freeze-susceptible, in which glycerol plays a crucial role in depressing supercooling point (SCP) to avoid the freezing injury. This study focused on a non-freezing injury classified into indirect chilling injury of S. exigua after a prolonged exposure to low temperatures much above SCPs. Exposure to 0 and 5°C for longer than 2weeks was lethal to all the immature stages. Among immature stages, eggs were the most susceptible to the low temperature treatments and pupae were the next susceptible. Among larvae, the third instar (L3) appeared to be more tolerant than the fifth instar (L5). The temperature treatment at 15°C allowed both L3 and L5 to exhibit a feeding behavior and induced little non-freezing injury, suggesting a minimal temperature threshold for optimal overwintering conditions of S. exigua. Three heat shock protein genes (Hsp70, Hsp74, Hsp83) were expressed in the larvae at the low temperature treatments. Only Hsp70 was inducible to the low temperatures in both L3 and L5 stages. RNA interference of Hsp70 expression led to significantly lose the survival rates of the treated larvae in the conditions inducing the non-freezing injury. These results suggest that Hsp70 plays a role in protecting S. exigua from the indirect chilling injury.

A Viral Histone H4 Joins to Eukaryotic Nucleosomes and Alters Host Gene Expression

ABSTRACT A viral histone H4 (CpBV-H4) is encoded in a polydnavirus, Cotesia plutellae bracovirus. Its predicted amino acid sequence is highly homologous to that of host insect histone H4 except for an extended N-terminal tail containing 38 amino acids with nine lysine residues. Its expression induces an immunosuppression of target insects by suppressing immune-associated genes, presumably through an epigenetic control. This study analyzed its molecular interaction with eukaryotic host nucleosomes and subsequent regulation of host gene expression. Purified recombinant CpBV-H4 could associate with nucleosomal components (H2A, H2B, H3, and H4) and form an octamer. Transient expression of CpBV-H4 in an insect, Tribolium castaneum, was performed by microinjection of a recombinant expression vector and confirmed by both reverse transcriptase PCR (RT-PCR) and immunoblotting assays. Under this transient expression condition, total RNAs were extracted and read by a deep-sequencing technique. Annotated transcripts were classified into different gene ontology (GO) categories and compared with those of control insects injected with a truncated CpBV-H4. Target genes manipulated by CpBV-H4 expression showing significant differences (fold changes > 109) included all GO categories, including development and immune-associated genes. When the target genes were physically mapped, they were found to be scattered on entire chromosomes of T. castaneum. In addition, chromatin immunoprecipitation against CpBV-H4 determined 16 nucleosome sites (P < 10−5) of the viral histone incorporation, which were noncoding regions near DNA-binding and inducible genes. These findings suggest that the viral histone H4 alters host gene expression by a direct molecular interaction with insect nucleosomes.

Protein tyrosine phosphatase encoded in Cotesia plutellae bracovirus suppresses a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella

Parasitization by an endoparasitoid wasp, Cotesia plutellae, inhibits a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella. This study tested an inhibitory effect of C. plutellae bracovirus (CpBV) on the metamorphosis of P. xylostella. Parasitized P. xylostella exhibited significantly reduced prothoracic gland (PTG) development at the last instar compared to nonparasitized larvae. Expression of the ecdysone receptor (EcR) was markedly suppressed during the last instar larvae parasitized by C. plutellae. By contrast, expression of the insulin receptor (InR) significantly increased in the parasitized larvae. Microinjection of CpBV significantly inhibited the larva-to-pupa metamorphosis of nonparasitized larvae in a dose-dependent manner. Injection of CpBV also inhibited the expression of the EcR and increased the expression of the InR. Individual CpBV segments were transiently expressed in its encoded genes in nonparasitized larvae and screened to determine antimetamorphic viral gene(s). Out of 21 CpBV segments, two viral segments (CpBV-S22 and CpBV-S27) were proved to inhibit larva-to-pupa metamorphosis by transient expression assay. RNA interference of each gene encoded in the viral segments was applied to determine antimetamorphic gene(s). Protein tyrosine phosphatase, early expressed gene, and four hypothetical genes were selected to be associated with the antimetamorphic activity of CpBV. These results suggest that antimetamorphosis of P. xylostella parasitized by C. plutellae is induced by inhibiting PTG development and subsequent ecdysteroid signaling with viral factors of CpBV.

In vivo transient expression for the functional analysis of polydnaviral genes.

Transient expression of a foreign gene in an organism is useful to determine its physiological function. This study introduces an efficient expression technique in the insect system using a recombinant eukaryotic expression vector. A recombinant construct expressing an enhanced green fluorescence protein (EGFP) gene under an immediately early promoter was injected into the larval hemocoel of Spodoptera exigua along with a cell transfection reagent. The expression of EGFP occurred earlier, and persisted for longer period with increasing injection dose. However, there was significant variation in expression efficiency among different cell transfection reagents. In addition, the transfection efficiency measured by RT-PCR varied among tissues with high expression of EGFP in hemocytes and fat body, but not in epidermis, gut, and nerve tissues. Two functional genes (CpBV15α and CpBV15β) derived from a polydnavirus were inserted into the eukaryotic expression vector and injected into S. exigua larvae. Expression levels in hemocytes and fat body were measured by RT-PCR and immunofluorescence assay. Both mRNAs and proteins were detected in the two tissues, in which expression signals depended on the amount of injected DNA. These immunosuppressive factors significantly inhibited hemocyte behavior, such as hemocyte-spreading, nodule formation, and phagocytosis. These results demonstrate the use of in vivo transient expression of polydnaviral genes for direct analysis of biological function in the host insect.

Transient expression of a viral histone H4 inhibits expression of cellular and humoral immune-associated genes in Tribolium castaneum.

A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4s, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. To analyze the epigenetic control activity of CpBV-H4 on expression of immune-associated genes, it was transiently expressed in larvae of Tribolium castaneum that had been annotated in the immune genes from a full genome sequence. Subsequent alteration of gene expression pattern was compared with that of its mutant form deleting N-terminal tail (truncated CpBV-H4). In response to bacterial challenge, T. castaneum induces expression of 13 antimicrobial peptide (AMP) genes. When CpBV-H4 was expressed, the larvae failed to express 12 inducible AMP genes. By contrast, when truncated CpBV-H4 was transiently expressed, all AMP genes were expressed. Hemocyte nodule formation was significantly impaired by expression of CpBV-H4, in which expressions of tyrosine hydroxylase and dihydroxyphenylalanine decarboxylase were suppressed. However, expression of truncated CpBV-H4 did not give any significant adverse effect on the cellular immunity. The immunosuppression of CpBV-H4 was further supported by its activity of enhancing bacterial pathogenicity of an entomopathogenic bacterium, Xenorhabdus nematophila, against larvae transiently expressing CpBV-H4. These results suggest that CpBV-H4 suppresses both humoral and cellular immune responses of T. castaneum by altering a normal epigenetic control of immune-associated gene expression.

JH modulates a cellular immunity of Tribolium castaneum in a Met-independent manner

Juvenile hormone (JH) regulates diverse physiological processes in insects during entire developmental stages. Especially, the identification of Methoprene-tolerant (Met), a JH nuclear receptor, allows us to better understand molecular actions of JH to control gene expressions related with metamorphosis. However, several physiological processes including cellular immune response and some molecular actions of JH have been suspected to be mediated via its non-genomic actions. To prove its non-genomic action, JH nuclear signals were suppressed by RNA interference (RNAi) of Met or its downstream gene, Krüppel homolog 1 (Kr-h1), in the red flour beetle, Tribolium castaneum. These RNAi-treated larvae failed to undergo a normal development and suffered precocious metamorphosis. Hemocytes of T. castaneum exhibited their spreading behavior on extracellular matrix and nodule formation in response to bacterial challenge. When the larvae were treated with either RNAi of Met or Kr-h1, the hemocytes of the treated larvae were responsive to JH without any significant difference with those of control larvae. These results suggest that the response of hemocytes to JH is not mediated by its nuclear signal. On the other hand, the JH modulation of hemocyte behaviors of T. castaneum was significantly influenced by membrane and cytosolic protein activities, in which ethoxyzolamide (a specific inhibitor of carbonic anhydrase), calphostin C (a specific inhibitor of protein kinase C) or ouabain (a specific inhibitor of Na(+)-K(+) ATPase) significantly suppressed the responsiveness of hemocytes to JH.

A viral factor, CpBV15α, interacts with a translation initiation factor, eIF2, to suppress host gene expression at a post-transcriptional level

An endoparasitoid wasp, Cotesia plutellae, possesses its specific symbiotic virus called C. plutellae bracovirus (CpBV) and parasitizes young larvae of Plutella xylostella. CpBV encodes CpBV15α, which was previously shown to interfere with host protein translation. In vivo transient expression of CpBV15α induced a significant decrease in a storage protein level without its transcriptional level change. In vitro translation assay using rabbit reticulocyte lysate showed that CpBV15α suppressed translation efficiency of mRNAs extracted from fat body of P. xylostella. Transient expression of CpBV15α in nonparasitized P. xylostella suppressed humoral immunity and development to pupal and adult stages. Immunoprecipitation (IP) of CpBV15α co-precipitated eIF2 and eIF2B (a guanine nucleotide exchange factor of eIF2) in parasitized P. xylostella. Additionally, IP of eIF2 co-precipitated CpBV15α as well as eIF2B and eIF5 in parasitized larvae. IP with eIF5 antibody showed that relative amount of eIF2 bound to eIF5 was much decreased in parasitized larvae, while significant amount of eIF2 was bound to CpBV15α. These results suggest that CpBV15α inhibits some host mRNA translation by sequestering eIF2.

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth, Plutellae xylostella

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect‐originated genes, but not with virus‐originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV‐E94k1, CpBV‐E94k2, CpBV‐E94k3, and CpBV‐E94k4. This study was conducted to predict the origin of CpBV‐E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV‐E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV‐E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.

Juvenile hormone regulates an expression of a late gene encoded in a polydnavirus, Cotesia plutellae bracovirus

An endoparasitoid wasp Cotesia plutellae encodes two host translation inhibitory factors (HTIFs) that are expressed in late larval stage of Plutella xylostella parasitized by C. plutellae. The late expressions of HTIFs seemed to be associated with decreasing titer of juvenile hormone (JH) at the last larval instar because an addition of pyriproxyfen (PYR, a JH analog) inhibited the late expression pattern of two HTIF genes. To understand their late expression control, promoter region of an HTIF gene called CpBV15α was cloned by inverse PCR. The cloned HTIF upstream region (1113 bp) possessed a putative JH response element (JHRE) and other promoter elements. The putative promoter region was rejoined with an open reading frame of enhanced green fluorescence protein (EGFP). When the recombinant vector construct was injected into early third instar larvae of nonparasitized P. xylostella, it was expressed in fourth larval instar at 72 h after injection, compared to relatively early expression in 24 h after injection of control construct containing a baculovirus immediate-early promoter. However, recombinant EGFP construct lost the late expression pattern when its promoter region was incomplete by truncating JHRE region. PYR application inhibited EGFP expression of the recombinant construct, but gave little influence on truncated constructs. Interestingly, when the complete promoter construct was injected to pupal stage, its late expression pattern was lost and showed early expression pattern. However, an addition of PYR to pupae, which had been injected with the complete promoter construct, inhibited the reporter gene expression. These results suggest that late expression of a HTIF (CpBV15α) is controlled by its promoter, which is sensitive to host JH titer.