Journal of Asia-Pacific Entomology 13 (2010) 313–318

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Journal of Asia-Pacific Entomology

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Transient expression of a viral histone H4, CpBV-H4, suppresses immune-associatedgenes of Plutella xylostella and Spodoptera exigua

Jaehyun Kim, Yonggyun Kim ⁎

Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea

⁎ Corresponding author.E-mail address: hosanna@andong.ac.kr (Y. Kim).

1226-8615/$ – see front matter © Korean Society of ApAll rights reserved.doi:10.1016/j.aspen.2010.05.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 March 2010Revised 3 May 2010Accepted 4 May 2010

Keywords:Cotesia plutellaePolydnavirusCpBVHistone H4CpBV-H4Immune

A viral histone H4, CpBV-H4, is encoded in the Cotesia plutellae bracovirus (CpBV) genome. This polydnavirusis symbiotic with C. plutellae, an endoparasitoid wasp. When the wasp parasitizes its host, Plutella xylostella,the symbiotic CpBV is delivered to host hemocoel and infects different internal tissues. CpBV-H4 encoded inthe virus exhibits high sequence similarity to host histone H4, except for an extended N-terminal tail (38amino acids long). When the CpBV-H4 cloned in a eukaryotic expression vector was transiently expressed inP. xylostella and a nonhost, Spodoptera exigua, it clearly inhibited several immune-associated genes, includingcecropin, gloverin, serpin, apolipophorin III, and transferrin. However, its truncated construct, prepared bydeleting 38 amino acids at the N-terminal tail, lost its inhibitory activity against immune-associated genes ofthe both species. This study has verified an inhibitory activity of CpBV-H4 against host immune-associatedgenes and has provided a possibility to expand its activity spectrum to the genes of other insect species.

© Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant ProtectionSociety, 2010 Published by Elsevier B.V. All rights reserved.

Introduction

Host immunosuppression is required for successful parasitizationby Ichneumonidae and Braconidae endoparasitoid wasps (Webb etal., 2000). These parasitoids use several factors derived from waspfemales or embryos to suppress both cellular and humoral immuneresponses of parasitized hosts (Webb and Strand, 2005). Maternalwasp factors include ovarian proteins, venom toxins, and poly-dnaviruses (PDVs), whereas embryonic wasp factors includeteratocytes and wasp larvae (Dahlman, 1991; Webb and Luckhart,1994). Though the relative contributions of these parasitic factors toinduce host immunosuppression vary, PDV is a main inducer withpersistent expression of its encoded genes (Beck et al., 2007). Theimportance of PDV in parasitism may come from an alternative viewon PDV as a wasp organelle system to deliver wasp genes tosuppress immune responses of the parasitized host (Federici andBigot, 2003).

Cotesia plutellae is, an endoparasitoid wasp of the diamondbackmoth, Plutella xylostella (Bae and Kim, 2004). It possesses a symbioticPDV, C. plutellae bracovirus (CpBV), which encodes more than 130

plied Entomology, Taiwan Entomol

putative genes (Kim et al., 2007). All known CpBV genes are able tosuppress cellular immune responses of P. xylostella (Kim, 2006). Forexample, the EPl-like CpBV gene suppresses total hemocyte popula-tion of P. xylostella, which results in significant reduction in noduleformation capacity in response to bacterial challenge (Kwon and Kim,2008). CpBV-15β is a secretory protein that can enter hemocytes toinhibit actin polymerization, which significantly impairs hemocyte-spreading behavior (Nalini et al., 2009). CpBV-IkB is a viral IkB thatpresumably inhibits a transcriptional factor, NFkB, which is involvedin expression of various immune-associated genes (Kim et al., 2006).Its expression significantly suppresses the antiviral response in P.xylostella (Bae and Kim, 2009).

CpBV-H4 is a viral histone H4 that is very similar to host H4 exceptfor 38 amino acids at the N-terminal region (Gad and Kim, 2008). Itsexpression occurs during the entire parasitization period and its proteinproducts are localized mostly in the nucleus of parasitized P. xylostellahemocytes (Gadet al., 2008).HistoneH4 is a componentof nucleosomesand is a unit for DNA condensation of eukaryotic chromosome (Wolffe,1992). Its long tail can be covalently modified to change chromatinstructure for gene expression control (Wu et al., 1986). Thus, it has beenspeculated that the prolonged N-terminal tail of the viral histone H4leads to aberrant control of host gene expression by altering chromatinstructure. CpBV-H4 expression suppressed expression of host histoneH4(GadandKim,2009). This study extended this observationby testinga hypothesis that CpBV-H4 could inhibit expression of host immune-

ogical Society and Malaysian Plant Protection Society, 2010 Published by Elsevier B.V.

314 J. Kim, Y. Kim / Journal of Asia-Pacific Entomology 13 (2010) 313–318

associated genes by its epigenetic control activity. Furthermore, thisstudy tested the inhibitory activity of CpBV-H4 against a non-naturalhost, Spodoptera exigua.

Materials and methods

Insects

P. xylostella larvae were reared under 25±1 °C and 16:8 h (L:D)photoperiod conditions with cabbage leaves. Adults were fed 40%sucrose. Late second instar larvae (4 days after oviposition at 25 °C)were parasitized by C. plutellae at 1:2 (wasp: host) ratio for 24 h underthe same rearing conditions. After emergence, adult wasps wereallowed to mate for 24 h and then used for experiments (Kim et al.,2006). Beet armyworm, S. exigua, originated from a field populationinfesting welsh onion in Andong, Korea. The larvae were reared onartificial diet (Gho et al., 1990) under the same rearing conditions.Adults were fed 10% sucrose.

cDNA construction

Total RNAs were extracted from different developmental stages ofnaive P. xylostella and S. exigua, and from the third instar larvae of bothspecies at different periods after immune challenge with Escherichiacoli (5×104 cells per larva). Trizol reagent (Invitrogen, Carlsbad, CA,USA) was used and total RNAs were precipitated with isopropanol.The RNA pellet was washed with 70% ethanol and resuspended indiethylpyrocarbonate-treated water. Total RNA (1 μg) was reverse-transcribed with RT-PCR premix (Intron, Daejeon, Korea).

RT-PCR analysis of immune-associated genes

The resulting single-stranded cDNA was used as template toamplify immune-associated genes using gene-specific primers(Table 1). The PCR reaction was performed in a total volume of 25 μl

Table 1RT-PCR primers for analysis of immune-associated genes of Plutella xylostella and Spodopte

Insect Genes Anneal

Plutella xylostella and Spodoptera exigua CpBV-H4 55

Truncated CpBV-H4 52

β -Actin 53

Plutella xylostella Transferrin (Tf) 52

Cecropin-A 53

Gloverin 52

Apolipophorin III (ApoLpIII) 53

Serpin-3 50

Spodoptera exigua Transferrin-1 (Tf-1) 52

Cecropin-A 53

Gloverin 55

Apolipophorin III (ApoLpIII) 53

Serpin-2 53

and run with 35 cycles of denaturation at 94 °C for 1 min, annealing atdifferent temperatures for different genes for 1 min, and extension at72 °C for 1 min, with a subsequent final extension at 72 °C for 10 min.

Transient expression of viral constructs

The recombinant CpBV-H4 (Gad and Kim, 2008) or truncatedCpBV-H4 (deleting 38 amino acid sequence in N-terminal region)(Gad and Kim, 2009) in the pIB expression vector (Invitrogen) wasused immediately for microinjection. The CpBV-H4 construct vectorwas injected into Nonparasitized third instar P. xylostella larvae wereinjected with 50 ng CpBV-H4 construct vector and nonparasitizedthird instar S. exigua larvae were injected with 500 ng CpBV-H4construct vector using a micro-injector (World Precision Instruments,Sarasota, FL, USA) equippedwith a glass capillary injection needle anda Micropipette puller PN-30 (Narishige, Tokyo, Japan) set at a rate of10 nl/s using PV830 Pneumatic Pump (World Precision Instruments).Microinjection was performed under a microscope (Olympus S730,Tokyo, Japan). The total RNA was extracted from these treated insectsand used to analyze transient expression of CpBV-H4 and truncatedCpBV-H4 (Table 1) by RT-PCR. β-Actin was used as a control.

Quantitative real-time PCR (qRT-PCR)

Using SYBR green dye (TOYOBO, Osaka, Japan), mRNA levels werequantified by real-time qRT-PCR using an Applied Biosystems 7500Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).Preparation of template cDNAs and primers performed as describedabove. The reactionmixture (20 μl) consisted of 10 μl of SYBRmixture,2 μl of each primer (2 pmol) and 6 μl of cDNA (90 ng). After a hot startat 94 °C for 10 min, the cycling reactions were conducted with 40cycles of 94 °C for 1 min, different annealing temperatures fordifferent genes for 1 min, and 72 °C for 1 min, with a final extensionat 72 °C for 10 min. β-Actin was used as an internal control withprimers. The comparative CT method (Livak and Schmittgen, 2001)

ra exigua in response to bacterial challenge.

ing temperature (°C) Primer sequence

5′- CGG GAT CCA TGG CTG ATC ATC CTA AAG G-3′5′- CGG AAT TCA CCT CCA TAA CCA TAG ATC A-3′5′-CGG GAT CCA TGG CAA GAG GAT TGG GCA AAG G-3′5′- CGG AAT TCA CCT CCA TAA CCA TAG ATC A-3′5′-ATG TAA CCC TGG TAT TGC TGA C-3′5′-GGA CGA TAG AGG GGC CAG AC-3′5′-ATC TGC GTA CCG TCT GAG TTC-3′5′-CTT CGG GTC AGG CGA CCA CTG-3′5′-ATG AAA CTG TCA AAT ATT-3′5′-TTT CCC AGT AGG TCT GG-3′5′-AGC ACT GAT GCG GGA GTA TT-3′5′-CTG TCT GAT CAT TCC CGC-35′-ATG GTC CGC CGC GAG GCG CC-3′5′-AGG CCT TCG GAG TTT CGG-3′5′-ATG TCA TTA GCA CTA TTT TTA ATA TTT TTT G-3′5′-TGC CAT TTT GAT TGT AGC AG-3′5′-GTC CCT CTC TGT CCT GAA GG-3′5′-CAG AAA CAC GAA GAA AGA TGG-3′5′-ATC GTT TAG CTT CGT GTT CGC-3′5′-GGA AAG AAA ATG GTG TGC CAA C-3′5′-CGT GGA CAT CTT CAG GGC C-3′5′-GTC GTG TTC AAT GCC ACC G-3′5′-ATG GTC GCC AAG TTG TTC GTG-3′5′-CTC CTG CGC GGT GTT CTG CA-3′5′-ATG GAT TCA AAG GCC CTC TC-3′5′-GCT GCT CCA GTG TTA AAG TC-3

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based on the CT value of β-actin gene was used to analyze the relativeexpression levels.

Data analysis

All studies were performed in three independent replicates andmeans±standard deviation were plotted using Sigma plot (Systatsoftware, Inc., Point Richmond, CA, USA). The means were comparedby a least squared difference (LSD) test of one-way ANOVA usingPROC GLM of SAS program (SAS Institute, 1989) and discriminated atType I error=0.05.

Results and discussion

Bacterial challenge induces expression of five different immune-associatedgenes in P. xylostella and S. exigua larvae

To test an inhibitory effect of CpBV-H4 on immune-associated genesin P. xylostella and S. exigua, the effect of selected genes on immuneresponse was first assessed in their expression (Fig. 1). Cecropin is an

Fig. 1. Inducible expression of selected immune-associated genes of Plutella xylostella (A)injected with Escherichia coli (5×104 cells/larva). The mRNA levels were analyzed by real-tiEach replication used 10 larvae. Different letters above the standard deviation bars indiidentification and PCR conditions are described in Table 1.

antimicrobial peptide which disrupts bacterial cell membranes, primar-ily of Gram-negative bacteria (Cheng et al., 2006). In P. xylostella, itsexpression was induced by bacterial challenge and its expression levelwas up-regulated as early as 4 h after E. coli injection. After the secondexpression peak at 24 h, the expression level declined to the basal level(Fig. 1A). In S. exigua, gene expression was up-regulated as early as 2 h(Fig. 1B).

Gloverin is an antimicrobial peptide against Gram-positive andGram-negative bacteria (Kaneko et al., 2007). Its expression was alsoinduced by bacteria challenge in both insect species. Compared tocecropin, its inducible expression occurred slowly. However, itsinducible intensity in P. xylostella was about five times higher thanthe control levels of both genes.

Serpins include factors for the negative control of prophenoloxidase(pPO) activation by inactivating serine proteinases that are involved inthe pPO activation pathway (Jiang and Kanost, 2000). In both species,serpin showed an acute up-regulation of its gene expression in responseto bacterial challenge.

Transferrin and apolipophorin III play roles in iron-binding and lipidtransport, respectively, and are associated with the immune response(Halwani et al., 2000; Yun et al., 2009). Their expressionswere inducible

and Spodoptera exigua (B) in response to bacterial challenge. Third instar larvae wereme quantitative RT-PCR. Each measurement was independently replicated three times.cate significant difference among means at Type I error=0.05 (LSD test). The gene

316 J. Kim, Y. Kim / Journal of Asia-Pacific Entomology 13 (2010) 313–318

and exhibited transient up-regulation in response to bacterial challengein both P. xylostella and S. exigua. Interestingly, the up-regulation oftransferrin expression delayed in P. xylostella with a transient decreaseat initial infection in S. exigua.

Transient expression of CpBV-H4 suppresses expression of immune-associated genes

The hypothesis that CpBV-H4 interrupts host immune-associatedgene expression was tested by its transient expression in nonpar-asitized larvae followed by bacterial challenge. As expected, all fiveimmune-associated genes were expressed in response to a bacterialchallenge in both P. xylostella (Fig. 2A) and S. exigua (Fig. 2B) controlgroups. Microinjection of a recombinant pIB construct containingCpBV-H4 induced gene expression in larvae of both species and theexpression of CpBV-H4 clearly suppressed the inducible expression ofthe five immune-associated genes in both insect species.

Fig. 2. Inhibitory effects of transiently expressed CpBV-H4 on expression of selectedimmune-associated genes of Plutella xylostella (A) and Spodoptera exigua (B) larvae.Third instar larvae of both species were injected with recombinant pIB constructscloned with CpBV-H4 or truncated CpBV-H4. After 24 h, the larvae were injected withEscherichia coli (5×104 cells/larva) and incubated at 25 °C. Control larvae were treatedwith the bacterial injection only. RNAswere collected and analyzed by RT-PCR using thegene-specific primers described in Table 1.

An extended N-terminal tail of CpBV-H4 is responsible for its inhibitoryactivity against expression of immune-associated genes

The N-terminal tail extension of CpBV-H4 possesses 38 aminoacids that are not found in histone H4 of P. xylostella (Gad and Kim,2008). It contains several lysine residues and may alter host geneexpression in its epigenetic control mode (Gad and Kim, 2009). Wetested this hypothesis using the expression of a truncated CpBV-H4construct prepared by deleting these 38 amino acids (Fig. 2).Microinjection of a recombinant pIB expression vector containingthe truncated CpBV-H4 to larvae of P. xylostella and S. exigua showedits expression in 24 h. The treated larvae showed clear expression ofthe five immune-associated genes and did not differ from the controllarvae.

A model of an epigenetic control of a viral histone H4, CpBV-H4, againsthost gene expression

Eukaryotic DNA is condensed from a diameter of 2 nm to 11 nmbyforming a series of nucleosome units and then become a 30 nmsolenoid chromatin structure by subsequent folding of nucleosomes(Turner, 1993). Condensed chromatin becomes resistant to access byRNA polymerase for gene expression. To be active in transcription,the nucleosomes should be released from at least the promoterregion of specific genes. This kind of chromatin remodeling is usuallymediated by epigenetic controls using covalent modification ofhistone proteins in which the N-terminal tail of histone H4 issubjected to covalent modifications, such as phosphorylation,acetylation, and methylation (Shogren-Knaak et al., 2006). Specifi-cally, several lysine residues of the N-terminal tail are used to bindDNA. However, their acetylation by histone acetyltransferase (HAT)loses their positive polarity, which causes dissociation of histonesfrom DNA, which can then be accessed by transcriptional factors orRNA polymerase (Peterson and Laniel, 2004).

This study demonstrated an inhibitory activity of CpBV-H4 againstimmune-associated genes in both P. xylostella and S. exigua.Considering the highly conserved nature of histone H4, we believethat CpBV-H4 may produce its inhibitory activity even in non-naturalhost (S. exigua). Here, we propose a model of the epigenetic control byCpBV-H4 on expression of immune-associated genes (Fig. 3). Immunechallenge may induce chromatin remodeling by removing nucleo-some structures in at least the promoter regions of immune-associated genes, which then become accessible to RNA polymerase(Fig. 3A). However, CpBV-H4-containing nucleosomes are not easilydetached from the DNA region due to the positive charge caused bythe increased number of lysines in the N-terminal extended tail of theviral H4 (Fig. 3B). Alternatively, the extended N-terminal tail mayprevent the host histone H4 from being acetylated by sequesteringHAT to CpBV-H4.

The immunosuppressive activity of CpBV-H4 caused by inhibitingthe expression of immune-associated genes may be useful inconstructing a biological agent. Kim et al. (2008) recombinedCpBV-H4 to a baculovirus and showed significant increase of theviral pathogenicity. However, it still needs to be determined howCpBV-H4 increased the viral pathogenicity because this studyshowed only its inhibitory activity against some antibacterial peptidegenes.

Acknowledgments

Most of the work was supported by AGENDA research programfunded by Rural Development of Administration, Korea. J. Kim wassupported by the second stage BK21 program of the Ministry ofEducation, Science and Technology, Korea.

Fig. 3. A model of epigenetic control of host immune-associated genes by a viral histone H4, CpBV-H4. Parasitized (P) and nonparasitized (NP) Plutella xylostella larvae arepresumed to be different in nucleosome structure due to the presence of CpBV-H4. (A) NP larvae exhibit inducible expression of immune-associated genes by immune challengevia chromatin remodeling that removes nucleosomes on the promoters. (B) P larvae may respond the immune challenge by stripping off the NP-nucleosomes on the promoter.However, P-nucleosomes reassociate with the open promoter to inhibit RNA polymerase access.

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