Institute of Cell Biology, University of Berne, Baltzerstrasse 4, CH-3012 Bern, Switzerland1
Author for correspondence: Beatrice Lanzrein. Fax +41 31 631 46 16. e-mail beatrice.lanzrein{at}izb.unibe.ch
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Abstract |
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Introduction |
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We are working with the polydnavirus of the braconid wasp Chelonus inanitus (CiV). C. inanitus is a solitary egg-larval parasitoid of Spodoptera littoralis. The egg stage is believed not to be immune competent (Salt, 1968 ) and thus the immediate early protection of the parasitoid egg at the hosts egg stage may not be important. Later on, however, once the host has become a larva, inhibition of encapsulation of the parasitoid larva by polydnaviruses appears to be essential (Pfister-Wilhelm & Lanzrein, 1996
; Stettler et al., 1998
). Parasitization by C. inanitus has a great influence on the development of S. littoralis as metamorphosis is induced precociously in the fifth instar followed by a developmental arrest in the prepupal stage (Grossniklaus-Bürgin et al., 1994
). The parasitoid larva in the presence of polydnavirus/venom has been shown to be responsible for the precocious onset of metamorphosis and to cause a premature decline in juvenile hormone (Pfister-Wilhelm & Lanzrein, 1996
; Steiner et al., 1999
). Polydnaviruses, synergized by venom, were shown to be responsible for the developmental arrest in the prepupal stage (Soller & Lanzrein, 1996
) and to cause an inhibition of the prothoracic gland and a reduction in haemolymph ecdysteroids at the stage of pupal cell formation (Grossniklaus-Bürgin et al., 1998
). The CiV genome is composed of at least 10 different DNA segments with sizes between 7 and 31 kbp (Albrecht et al., 1994
). Four segments (CiV12, CiV14, CiV14.5 and CiV16.8) have been sequenced completely and for CiV12 and CiV14 the integration/excision site in the wasp genome has been characterized (Gruber et al., 1996
; Wyder et al., 2002
). Northern dot blots with total CiV DNA or segments CiV12, CiV14 and CiV16.8 as probe revealed the presence of viral transcripts throughout the time-course of parasitization; but levels were very low and mainly increased in the last larval instar, in particular in the case of CiV12 and CiV14 (Johner et al., 1999
).
Here we present data for the first CiV genes to be cloned and characterized. As both are situated on segment CiV14 they are named CiV14g1 and CiV14g2. The cDNA of CiV14g1 has a size of 2036 bp and encodes a predicted protein of 548 amino acids; the cDNA of CiV14g2 has a size of 618 bp and encodes a predicted protein of 77 amino acids. Transcript quantities of both genes are low up to the penultimate instar and increase in the hosts last instar at the stage of pupal cell formation.
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Methods |
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Total RNA isolation.
RNA from first to fourth instar S. littoralis was isolated as described by Johner et al. (1999) , but with minor modifications. The purification of the homogenate by the QIAshredder was replaced by a digestion with proteinase K. Briefly, DEPC H2O and proteinase K (10 mg/ml) were added to the homogenate in the ratio of 1·65:0·35:1 (by vol.), incubated for 1·5 h at 45 °C and centrifuged for 10 min at 11000 g. The supernatant was then mixed with an equal volume of lysis buffer RLT (Qiagen) and an equal volume of ice-cold ethanol and applied onto RNeasy mini spin columns (Qiagen). For isolation of RNA from fifth and sixth instar larvae the RNeasy Midi Kit (Qiagen) was used. Larvae were homogenized with a Polytron PT10-35/PTA 10s (Kinematica) in lysis buffer RLT (0·45 ml per 100 mg tissue) containing 1% (v/v)
-mercaptoethanol. The proteinase K digestion of the homogenates was done as described above and the subsequent steps were performed according to the RNeasy Midi Kit protocol, including the DNase digestion on the column. After elution a second DNA digestion was carried out as described by Johner et al. (1999)
.
cDNA library construction.
RNA from either 10-day-old feeding fifth instar parasitized larvae (=L5p library) or X-ray-parasitized 12-day-old sixth instar larvae in the early pupal cell formation stage (=XL6 library) was isolated as described above. For poly(A)+ mRNA isolation the PolyATract mRNA Isolation System (Promega) protocol for small scale mRNA isolation was used. cDNA libraries were constructed following the SMART PCR cDNA Library Construction Kit (Clontech) protocol. cDNAs were ligated into vector gt11 and packaged into E. coli Y1090r- with the Gigapack III Gold Packaging Extract (Stratagene), following the manufacturers instructions. The unamplified L5p library contained 1·35x106 clones with a recombination efficiency of 44% and the unamplified XL6 library contained 0·62x106 clones with a recombination efficiency of 80%. The two libraries were then amplified according to the Clontech protocol and yielded titres of 5·5x1010 p.f.u./ml (L5p) and 3·9x1010 p.f.u./ml (XL6).
Screening probes.
On the basis of ORF predictions on CiV14 (Wisconsin Package Version 10.1, Genetic Computer Group) two pairs of primers were designed, namely CiV14U3A/CiV14L3A and CiV14U4A/CiV14L4A, resulting in products of 193 bp and 157 bp, respectively (Fig. 1a and accession no. AJ278677). The 5000 bp HindIII fragment of CiV14 was purified by gel extraction (QIAEX II Gel Extraction Kit, Qiagen) and used as a template. The PCRs were carried out in a volume of 50 µl with 107 molecules of template, 0·3 µM of each primer, 2·6 U Expand High Fidelity Enzyme-Mix and 1x PCR DIG Probe Synthesis Mix (containing 70 µM DIG-11-dUTP). The cycling conditions were: 5 min at 95 °C; 35 cycles of 95 °C, 1 min; 54 °C, 1 min; 68 °C, 3 min; and a final elongation step at 68 °C for 10 min. PCR products were analysed on a 1% agarose gel and the amount of DNA containing DIG-11-dUTP was determined by comparison with a DIG-labelled control DNA (Roche). In addition DIG-High Prime- (Boehringer Mannheim) labelled CiV DNA was used as a probe (Johner et al., 1999
). Subsequently, the following screening probes were made: a CiV14g1-specific probe with primers CiV14g1U2/CiV14g1L2 (Fig. 1a
and accession no. AJ278677) with a length of 160 bp and two CiV14g2-specific probes with primers CiV14g2U2/CiV14g2L2 and CiV14g2U3/CiV14g2L3 (Fig. 1b
and accession no. AJ278677) with a length of 252 bp and 313 bp, respectively.
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DNA cloning and sequencing.
DNA of clones
6/4.1,
6/5.1 and
6/6.5 was isolated with the Qiagen Lambda Maxi Kit and digested with EcoRI, NotI and SalI, alone or in combination. All three restriction sites are present in the SMART adapter ligated on both sides of the cDNA. As no NotI restriction site was detected in the inserts by analysis of the digested DNA on a 0·8% agarose gel, cDNA inserts were finally cut-out with a NotI digestion, purified by gel extraction (QIAEX II Gel Extraction Kit, Qiagen) and cloned into pBluescript II KS(+) vectors. The insert of clone
6/7.3 was amplified by PCR using
gt11 forward and reverse primers (Galas et al., 1980
) with the Taq PCR core kit (Qiagen) in a volume of 50 µl, consisting of 12·5 pmol of each primer, 200 µM of each dNTP, 2·5 U Taq polymerase and the DNA template (1 µl of phage lysate). Cycling was as follows: 95 °C, 6 min; 30 cycles of 95 °C, 1 min; 48 °C, 1 min; 74 °C, 3 min; and a final extension step at 74 °C for 10 min. After analysing the product on a 1·2% agarose gel the DNA fragment with a size of about 800 bp was cloned into the vector pCR 2.1-TOPO according to the TOPO TA Cloning Kit (Invitrogen) protocol. Plasmid DNA was isolated using Wizard Plus SV Minipreps (Promega) as specified by the manufacturer, followed by ethanol precipitation. Sequencing was done with the Thermo Sequenase Sequencing Kit (Amersham) with IRD800 labelled primers. PCR conditions were: 94 °C, 5 min; 30 cycles of 94 °C, 30 sec; 55 °C, 45 sec; 68 °C, 2 min; and a final elongation step at 68 °C for 10 min. Automatic sequencing was carried out on a Gene ReadIR 4200 (Licor).
Northern blot analysis.
Total RNA was denatured in 50% (v/v) DMSO, 4% (v/v) deionized glyoxal and 10 mM sodium phosphate, pH 6·85, for 20 min at 50 °C and separated on an agarose gel (1·2 g agarose/100 ml 10 mM sodium phosphate). As a marker, RNA Molecular Mass Marker I (0·36·9 kb) (Roche) was used. Transfer to positively charged nylon membranes (Roche) was by capillary forces in 20xSSC (1xSSC=150 mM NaCl, 15 mM sodium citrate, pH 7·0) as described by Sambrook et al. (1989) . UV-cross-linking was as described above and membranes were prehybridized for at least 5 h in 6xSSC, 5x Denhardts solution (1xDenhardts solution=0·2 g/l Ficoll 400, 0·2 g/l polyvinylpyrrolidone, 0·2 g/l BSA), 1 g/l SDS and 100 µg/ml denatured herring sperm DNA at 60 °C. The blots were hybridized at 60 °C for 2 days in the same buffer with an [
-32P]dCTP-labelled DNA probe (approximately 106 c.p.m./ml hybridization solution). Labelling was done with the Ready To Go Kit according to the manufacturer (Amersham) whereby 1550 ng DNA served as template. Finally, the blots were washed 3x20 min in 1xSSC containing 0·1 g/l SDS at 60 °C, exposed to a PhosphorScreen and analysed on a PhosphorImager (Molecular Dynamics).
Southern blot analysis.
Calyx fluid collection from female reproductive tracts of C. inanitus and DNA isolation was as described (Albrecht et al., 1994 ). Undigested and HindIII-digested calyx DNA and HindIII-digested clone 2A6, which contains the entire CiV14 segment, were separated on a 0·8% agarose gel by field inversion gel electrophoresis (FIGE). Electrophoresis as well as blotting, probe labelling and high stringency washes were as described by Wyder et al. (2002)
.
3'RACE (rapid amplification of cDNA ends).
To identify the 3' end of CiV14g1, a 3'RACE was done. Poly(A)+ mRNA was isolated from X-ray-parasitized sixth instar S. littoralis larvae in the early cell formation stage and reverse transcribed with the Superscript First-Strand Synthesis System for RTPCR (Life Technologies), with the supplied oligo(dT) primer replaced by a modified primer (5' GACTCGAGTCGACATCGAT17 3'). The cDNA was precipitated with isopropanol and the dried DNA pellet was dissolved in 10 mM TrisHCl, pH 8·0. The PCR reaction was performed with the Taq PCR Core Kit (Qiagen) in presence of 50 pmol primer (5'GACTCGAGTCGACATCG 3') and 50 pmol of the CiV14g1-specific primer CiV14g1.R (Fig. 1a and accession no. AJ278677). Cycling conditions were: 94 °C, 5 min; 40 cycles of 94 °C, 1 min; 65 °C, 1 min; 74 °C, 2 min; and final elongation at 74 °C for 10 min. This led to a product of about 650 bp which was then gel extracted (QIAEX II Gel Extraction Kit, Qiagen) and cloned into the vector pCR 2.1-TOPO as described above.
Real-time PCR.
Total RNAs from parasitized, X-ray-parasitized or nonparasitized S. littoralis were reverse transcribed with the Superscript First-Strand Synthesis System for RTPCR (Life technologies), with 15 µg template was used with oligo(dT) as primer. For the subsequent real-time PCR primers were designed with Primer Express prerelease version 1.0 (PE Applied Biosystems). The primer pairs for CiV14g1 and CiV14g2 were CiV14g1U1/CiV14g1L1 and CiV14g2U1/CiV14g2L1 (Fig. 1a, b
and accession no. AJ278677); both produce an amplicon of 51 bp. As an endogenous standard a primer pair specific for S. littoralis
-actin, namely actin forward and actin reverse (accession no. Z46873), was used. The PCR was performed with the SYBR Green I Reaction System (Eurogentec); briefly, an appropriate amount of cDNA was amplified in a volume of 30 µl containing 200 µM each dNTP, 3·5 mM MgCl2, 0·75 U Hot GoldStar enzyme, 200 nM of each primer and 0·9 µl of a 1/2000 dilution of Sybr Green I stock. Cycling conditions were: 95 °C, 10 min; followed by 40 cycles of 95 °C, 15 s; and 60 °C, 1 min. Measurement was done in 96-well Optical Reaction Plates (Applied Biosystems) with the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). The quality of primers was tested by analysis of the melting curves and by analysis of the PCR products on a 3·5% MetaPhor agarose gel.
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Results |
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A comparison of CiV14g1 and CiV14g2 cDNAs with genes predicted on the genomic sequence using Fgenesh (Solovyev & Salamov, 1999 ) with either Drosophila or Human settings is shown in Fig. 3
. In the case of CiV14g1, 100% of the coding nucleotides were predicted with Human settings compared to 66% with Drosophila settings, but Human settings gave 21% of false positives as compared to 7% with Drosophila settings. CiV14g2 was not predicted with Human settings, but with Drosophila 56% of the coding nucleotides were correctly predicted. Fgenesh also predicted a third gene on CiV14, but its existence is uncertain as real-time PCRs with parasitized and X-ray-parasitized larvae were inconclusive (data not shown). It remains possible that this gene exists but is either transcribed at a very low level in parasitized and X-ray-parasitized larvae or only in the wasp.
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Discussion |
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CiV14g1 and CiV14g2 are both localized on CiV14 and a larger segment (Fig. 6). As additional probes of CiV14 also hybridized to the same larger segment (Wyder et al., 2002
) it appears that the entire CiV14 is also present nested in another larger segment. In three braconid larval parasitoids identified, viral cDNAs hybridized to a single segment only (Harwood et al., 1994
; Yamanaka et al., 1996
; Strand et al., 1997
; Trudeau et al., 2000
) and in one case to several segments (Varricchio et al., 1999
). In the ichneumonid Campoletis sonorensis some cDNAs hybridized to one segment only and others, encoding the abundantly expressed Cys-motif genes, hybridized to several segments which were nested. It was thus proposed that nested segments are likely to encode abundantly expressed genes (Webb & Cui, 1998
).
Expression of CiV14g1 and CiV14g2 in the course of host development and comparison with larval parasitoids
CiV14g1 and CiV14g2 were both found in the XL6 library, which was made from mRNA of X-ray-parasitized sixth instar larvae in the early pupal cell formation stage. Measurement of transcript quantities of CiV14g1 and CiV14g2 in parasitized and X-ray-parasitized larvae from the first to the last instar showed that expression of both genes was highest in X-ray-parasitized sixth instar larvae at the early pupal cell formation stage (Figs 7b, c
). In parasitized larvae, the latest stage investigated (L5p) was late feeding in the fifth (=last) instar, because later on the parasitoid has grown enormously and occupies a large portion of the hosts body (Grossniklaus-Bürgin et al., 1994
). The developmental stage L5p corresponds to that of late feeding sixth instar X-ray-parasitized larvae and quantities of transcripts are very similar in L5p and XL6fl (Figs 7b
, c
). Thus, only the initial increase in CiV14g2 transcripts can be seen in parasitized larvae (Fig. 7c
). Expression of this gene is strongly upregulated in X-ray-parasitized sixth instar larvae at the stage from late feeding to early pupal cell formation (Figs 5
and 7c
). This is precisely the time-point when prothoracic gland activity and ecdysteroid titres are reduced in X-ray-parasitized larvae as compared to nonparasitized larvae (Grossniklaus-Bürgin et al., 1998
; Lanzrein et al., 2001
). Also, transcription of CiV14g1 is upregulated at the stage of pupal cell formation but to a lesser extent than CiV14g2 (Figs 7b
, c
) and total quantities of transcripts appear also to be lower for CiV14g1 than CiV14g2 as deduced from the Northern blots (Figs 4
and 5
). This suggests that both genes might be involved in suppressing host ecdysteroids at this stage.
Our data are the first to show strong upregulation of polydnavirus genes towards the end of parasitization. All other data on the temporal appearance of polydnavirus transcripts stem from larval parasitoids where evasion or immediate suppression of the hosts immune system is required to prevent encapsulation of the parasitoid egg. In hosts of several braconids, viral transcripts with sizes between 0·4 and 2·0 kb have been identified and they appeared as early as 30 min after parasitization and reached highest levels 124 h after parasitization (Harwood & Beckage, 1994 ; Asgari et al., 1996
; Yamanaka et al., 1996
) or injection of calyx fluid/venom (Strand et al., 1997
; Trudeau et al., 2000
). Most of these transcripts were found in host haemocytes, but other tissues have also been reported to contain viral transcripts, e.g. fat body, midgut, Malpighian tubules, nervous tissue and the prothoracic gland (Strand et al., 1992
; Harwood & Beckage, 1994
; Harwood et al., 1994
; Asgari et al., 1996
; Varricchio et al., 1999
). In the egg-larval parasitoid C. inanitus the site of polydnavirus transcription has not yet been analysed in detail, but Northern dot blots with CiV14 as a probe indicate transcription in the haemolymph of parasitized fifth instar larvae (Johner et al., 1999
). As CiV14g1 and CiV14g2 are the major genes transcribed from CiV14 (see above) and as Northern dot blots with subclones of CiV14, which encompass CiV14g1 or CiV14g2, gave clear signals with haemolymph (data not shown), it appears that both genes are transcribed in haemocytes. Their expression in other tissues is currently being investigated.
Among the ichneumonids investigated up to now most information is available for C. sonorensis and its host Heliothis virescens. In this host, viral genes are expressed rapidly and persistently throughout endoparasitoid development with little significant variation in mRNA levels from 4 h to 8 days after parasitization; for the Cys-motif genes a role in suppression of the host immune reaction has been documented (Blissard et al., 1987 ; Theilmann & Summers, 1988
; Cui & Webb, 1996
; Webb & Cui, 1998
; reviewed in Webb, 1998
). In Choristoneura fumiferana parasitized by Tranosema rostrale a 650 bp transcript was detected from 1 day after parasitization onwards with a maximum at 34 days post-parasitization; highest quantities of transcripts were found in the cuticle and lower amounts in the fat body, midgut and haemolymph (Béliveau et al., 2000
). In haemocytes of S. littoralis parasitized by Hyposoter didymator three polydnavirus-specific mRNAs were seen from 2 h onwards (Volkoff et al., 1999
).
On Northern blots with 20 µg total RNA from parasitized S. littoralis and CiV DNA as probe we could never see signals (data not shown) whereas in hosts of several larval parasitoids strong signals were seen under similar conditions (Asgari et al., 1996 ; Varricchio et al., 1999
; Volkoff et al., 1999
; Béliveau et al., 2000
). It thus appears that the level of viral gene expression is generally lower in this egg-larval parasitoid as compared to larval parasitoids. Interestingly, the quantitiy of viral DNA injected per egg is also much lower, approximately 0·1 ng (Lanzrein et al., 2001
) as compared to 2·4 and 6·5 ng for the two larval parasitoids investigated (Theilmann & Summers, 1986
; Strand et al., 1992
). Additional analyses with other egg-larval parasitoids would be necessary to find out whether the intensity and pattern of expression of polydnavirus genes is generally different between egg-larval and larval parasitoids or whether the observations reported here are specific for the C. inanitusS. littoralis system.
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Acknowledgments |
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Footnotes |
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References |
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Received 27 September 2001;
accepted 10 January 2002.