Winged bean chymotrypsin inhibitors retard growth of Helicoverpa armigera☆

Manasi A. Telang a,b, Ashok P. Giri a,b, Prashant S. Pyati b, Vidya S. Gupta b,⁎,Mechthild Tegeder a, Vincent R. Franceschi aa School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USAb Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 13 August 2008Received in revised form 15 October 2008Accepted 23 October 2008Available online 11 November 2008

Received by A. Bernardi

Keywords:Psophocarpus tetragonolobusOrgan-specific mRNA expressionKunitz-type proteinase inhibitorDigestive gut proteinasesAntimetabolic effect

Two putative Kunitz-type chymotrypsin inhibitor genes (WCI2 and WCI5) were isolated from winged bean(Psophocarpus tetragonolobus (L.) DC). While WCI2 has previously been characterized, WCI5 represents a newmember of the WCI family. WCI5 was exclusively expressed in winged bean seeds. Theoretical translation ofboth the genes resulted into polypeptides of 207 amino acids with 86% sequence similarity. The polypeptidesequences contain four half-cysteine residues and a well-conserved Leu65–Ser66 reactive site, typical forchymotrypsin inhibitors. WCI5 and WCI2 were expressed in Pichia pastoris and the recombinant proteinswere assayed against various proteinases. Both the inhibitors strongly inhibited commercially availablebovine chymotrypsin. More importantly, gut proteinases of Helicoverpa armigera larvae that damage manyimportant crop plants, were inhibited by WCI2 and WCI5. In addition, both proteinase inhibitorsdemonstrated significant reduction of growth of H. armigera larvae after feeding on inhibitor incorporatedartificial diets. The inhibitory effects of WCI2 and WCI5 on activity of proteinases and larval growth makethese proteins and their genes promising candidates for enhancing plant defense against H. armigera usingtransgenic plants.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Winged bean (Psophocarpus tetragonolobus (L) DC) is a tropicallegume with high protein and oil content and has attracted attentionas a protein food source (Peyachoknagul et al., 1989; Habu et al., 1992).In addition to seeds, winged bean has tuberous root as a propagativeorgan and both seeds and root contain proteinase inhibitors (PIs),including chymotrypsin and trypsin inhibitors. The concentration ofchymotrypsin inhibitors in these organs is higher than that of thetrypsin inhibitors. Many leguminous seeds contain two major types ofPIs, Bowman–Birk (8 kDa) and Kunitz (20 kDa) family. Winged beanseeds have been reported to contain at least three Bowman–Birk-typeinhibitors (BBI) and nine Kunitz-type trypsin or chymotrypsininhibitors (Peyachoknagul et al., 1989; Giri et al., 2003). We have

previously reported that the BBIs are thermostable but get inactivatedby reducing agents, while the Kunitz PIs can be inactivated at 90 °Cand are stable towards the reducing agents. Moreover, Kunitz-type PIsstrongly inhibits the gut proteinases ofHelicoverpa armigera comparedto the BBIs (Giri et al., 2003).

Four Kunitz chymotrypsin inhibitors fromwinged bean seeds (WCI1to WCI4) have been purified earlier, of which WCI2 and WCI3 werereported to account for about 22 and 72% of the seedWCIs, respectively(Habu et al., 1992). A cDNA encoding WCI3 has been isolated anddevelopmental regulation of WCI transcripts has been demonstratedduring seedmaturationperiod (Habu et al.,1992). Small amounts of theWCI3 protein andmRNA have also been detected inwinged bean stem,where it was localized in sieve tubes. This supports the possibleinvolvement of Kunitz PIs in defense against insects that suck thephloem sap or against bacteria that invade uponwounding (Habu et al.,1996). While WCI3 has been analyzed extensively with respect to itsphysicochemical, physiological and structural properties, informationabout other inhibitors in winged bean is limited (Datta et al., 2001).

Although putative winged bean trypsin/chymotrypsin inhibitorgenes have been characterized earlier (Peyachoknagul et al., 1989;Habu et al., 1992; Ghosh and Singh, 1997) potential of their encodedproteins to inhibit insect gut proteinases has not been demonstratedso far. Our earlier work established winged bean PIs as activityinhibitors of midgut proteinases of H. armigera. In the present study,isolation of two putative winged bean chymotrypsin inhibitor genes(WCI2 andWCI5), analysis of their expression in different plant organs

Gene 431 (2009) 80–85

Abbreviations: BAρNA, N-α Benzoyl-DL-Arginine ρ-nitroanilide; BBI, Bowman–BirkInhibitors; CBB R250, Coomassie Brilliant Blue R-250; HGP, Helicoverpa armigera GutProteinases; PAGE, Polyacrylamide Gel Electrophoresis; PCR, Polymerase ChainReaction; PI, Proteinase Inhibitor; WCI, Psophocarpus tetragonolobus ChymotrypsinInhibitor; RT-PCR, Reverse Transcriptase PCR; SDS, Sodium Dodecyl Sulfate; WCI,Winged bean Chymotrypsin Inhibitor.☆ This publication is dedicated to our friend and colleague, Vincent Franceschi(1953-2005), who played a key role in the McKnight Foundation sponsoredCollaborative Crop Research Program between India and the USA. He is greatly missed.⁎ Corresponding author. Plant Molecular Biology Unit, Division of Biochemical

Sciences, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008 (M. S.),India. Tel.: +91 20 2590 2247; fax: +91 20 2590 2648.

E-mail address: vs.gupta@ncl.res.in (V.S. Gupta).

0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2008.10.026

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and recombinant gene expression in Pichia pastoris is presented.While WCI2 has been described previously (Peyachoknagul et al.,1989; Habu et al., 1992), this work reports a new gene WCI5. Theabbreviation means fifth chymotrypsin inhibitor reported from theplant P. tetragonolobus as against WCI1 to WCI4 (winged beanchymotrypsin inhibitors 1 to 4). Antagonistic effects of recombinantWCI2 andWCI5 on H. armigera are described using both in vitro and invivo assays.

2. Materials and methods

2.1. Materials

Seeds of winged bean cultivar iiHP Sel 21 were obtained fromPunjabrao Agricultural University, Akola, India. Plants were grown in agrowth chamber at 24 °C, 65% humidity and 12 h light with 2 hramping. Fifth instar larvae of H. armigera were collected fromchickpea fields or reared in the laboratory on an artificial diet withor without added winged bean PIs (Harsulkar et al., 1999).

2.2. Isolation of WCI genes

Genomic DNAwas extracted fromyoungwinged bean leaves by theCTAB method (Rogers and Bendich, 1988) and used as a template forWCI gene isolation by polymerase chain reaction (PCR) approachsinceWCIs are predicted to lack introns (Habu et al.,1992). The primerswere designed along the flanking regions of known Kunitz-typewinged bean WCIs sequences (D31703 and S96735, Table 1S). Twodifferent primer pairs were used: (1) 5′-CCAgACTATTTACAAATTCg-3′and 5′-gCATTCATTTAgTCATAAgC-3′ and (2) 5′-ggTgAgTAgAAAA-gAAACCC-3′ and 5′-gCACACATgATTTTTAAg-3′. PCR was performedusing standard conditions and Accumprime pfx DNA polymerase(Invitrogen, USA). Three independent reactions were carried out witheach primer pair to ensure error-free sequences. The PCR productswere cloned into pGEM-T Easy vector (Promega, USA) and at least threeclones per PCR reaction were sequenced. After ensuring sequencefidelity they were compared to each other and publicly availablesequences by NCBI BLAST search (http://www.ncbi.nlm.nih.gov) andanalyzed by sequence alignment (DNASTAR, USA), and phylogenetictree position (PAUP⁎4.0b10 program; Swofford, 2001).

2.3. Analysis of tissue-speci!c expression of WCIs

For analysis of WCI expression in winged bean plant organs, totalRNA was isolated from roots, leaves, stems, flowers, pod walls, seedcoats, developing seeds (40 days after flowering; 40 DAF) and matureseeds (90 DAF) of winged bean (3-month-old plants) according toPélissier et al. (2004). Total RNA (1 !g) was used for semi-quantitativereverse transcriptase polymerase chain reaction (RT-PCR). First strandcDNA synthesis was carried out with M-MLV reverse transcriptase(Invitrogen, USA) at 37 °C for 50 min and PCR was performed understandard conditions with primers; 5′-ATgAAgAgTACTACATTTC-3′ and5′-TTAATggCTTgATgCAgTT-3′ for WCI2 and 5′-ATgAAgAgTACCATA-TTTC-3′ and 5′-TTAATggCTTgATgCAgTT-3′ for WCI5, respectively. Forcontrol of even amounts of RNA in each tissue sample, 18S rRNA wasamplified with primers 5′-ATTCTATgggTggTggTgC-3′ and 5′-CCATC-CAATCggTAggAgC-3′.

2.4. Expression of proteinase inhibitors in P. pastoris

For cloning into the P. pastoris expression vector pPIC9 (Invitrogen,USA), the newly isolated WCI2 and WCI5 were amplified by PCR usingthe following primers: 5′-AAAAAACTCgAgAAAAgAgAggCTgAAgCTgA-TgATgATTTggTC-3′ and 5′-AAAAAAgCggCCgCTTAATggCTTgATgCAgTT-3′ forWCI2 and 5′-AAAAAACTCgAgAAAAgAgAggCTgAAgCTgATCATgA-TTTggTC-3′ and 5′-AAAAAAgCggCCgCggATCAgTAgTgCTTAATggCT-3′

for WCI5. XhoI and NotI restriction sites (underlined) were added,respectively. PCRwas carried outwith PfuDNApolymerase (Fermentas,USA) under standard conditions and the resulting DNA fragments werecloned into pPIC9 and sequenced. pPIC9/WCI2 and pPIC9/WCI5 (each3 !g) were linearized with SalI, transferred into P. pastoris strain GS 115by PEG 1000 method. The transformed cells were selected andconfirmed to express the recombinant WCIs by following themanufacturer's protocol (Invitrogen, USA) and the X-ray film contactprint method (Pichare and Kachole, 1994). The X-ray film contact printmethod involved electrophoretic separation of concentrated proteinsfrom the culture supernatant (50 !l) on 12% native polyacrylamide gel.After electrophoresis, the gel was equilibrated in 0.1 M Tris–HCl bufferand incubated in 0.1% chymotrypsin solution. The gel was thenoverlaid on an X-ray film taking care that no air bubble wasintroduced between the gel and the film and kept for 3–5 min. Thefilm was washed with water and inhibitor activity bands werevisualized as zones of unhydrolyzed gelatin on X-ray film. The X-rayfilms were developed using Kodak 163 DA developer and thencontact-printed to obtain the photographs. Yeast clones expressinghigh amounts of inhibitor protein were taken for large-scaleexpression of the WCIs by scaling up (Invitrogen, USA). Supernatantwas harvested after completion of the fermentation process viacentrifugation, and NaCl was added to a final concentration of 2 M.The entire volume was loaded on a Phenyl-Sepharose column(hydrophobic interaction chromatography) pre-equilibrated with2 M NaCl in de-ionized water. The expressed protein was then elutedunder a reverse NaCl gradient (2.0–0.0 M). Fermentation was repeatedto obtain sufficient quantity of expressed proteins. The homogeneityof the recombinant protein was analyzed by SDS-PAGE, freeze-driedand stored at 4 °C until use in further assays.

2.5. Preparation of HGP and winged bean seed extracts

Larvae of H. armigera were collected from chickpea, pigeon peaand okra fields or grown in the laboratory (Harsulkar et al., 1999;Telang et al., 2003). Midguts from fifth instar H. armigera larvae wereremoved by dissecting the larvaemid-dorsally under sterile conditionsand guts were stored at !20 °C until further use. For protein extraction,the gut tissuewas mixed with (w/v; 1:1) of 0.2 M glycine-NaOH buffer(pH 10) and allowed to stand for 2 h at 4 °C. The gut luminal contentwas removed by centrifugation at 14,230 ×g for 15 min at 4 °C. Theresulting supernatantwas analyzed for proteinase activity. Drywingedbean seeds were ground to a fine powder, and defatted anddepigmented; and seed extract was prepared as described earlier(Harsulkar et al., 1999).

2.6. Proteinase and proteinase inhibitor assays

Inhibition potential of WCI2 and WCI5 against trypsin, chymo-trypsin and HGPwas analyzed by an azocaseinolytic assay (Brock et al.,1982) as described earlier (Patankar et al., 2001). HGPs were isolatedfrom larvae fed on host plants okra (ok), pigeon pea (pp) andchickpea (cp) or fed with artificial diet as control or artificial dietsupplemented with PIs from winged bean seed (wb), Momordicacharantia trypsin inhibitor (McTI-II, P10295: 20 !g/g of artificial diet)and potato inhibitor II (PIN-II; CAA27730: 3 trypsin inhibitor units/gof artificial diet as described by Harsulkar et al., 1999). The syntheticsubstrate sulphanilamide-azocasein was used for assaying totalproteolytic activities of trypsin, chymotrypsin, and the differentHGPs. One unit of enzyme was defined as the amount of proteinaserequired for increase in absorbance by 1 OD at 450 nm in 30 min dueto liberation of sulphanilamide on proteolysis of substrate in 1 mlreaction volume. 0.4 U of each proteinase (1.2 !g trypsin, 2.4 !gchymotrypsin and 1–5 !l of HGP extracts) was incubated withincreasing amounts of WCIs (0.1–5.0 !g) for 15 min at 25 °C and thenresidual proteolytic activity was assayed.

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2.7. H. armigera feeding studies

Effects of WCI2 and WCI5 on growth and development of H.armigera were investigated by larval feeding assays. The artificial dietas suggested by Nagarkatti and Prakash (1974) for H. armigera wasmodified for our laboratory conditions as described earlier (Guptaet al., 2000). The amount of WCI2 per gram diet (1-fold concentration)required for maximum inhibition of total HGP from a single larva atfifth instar was calculated by x=(T⁎ I) /E, where x=amount of inhibitorper gramdiet, T=total volume of HGP from one insect gut, I=minimumamount (!l) of inhibitor (at a concentration of 1 !g/!l) required formaximum inhibition of 0.4 U of HGP and E=volume (!l) of HGPcorresponding to 0.4 U. For the actual feeding assay, 20 !g of WCI2 pergram artificial diet equivalent to 0.25-fold concentration was selectedbased on pilot experiment. Similarly, 20 !g of WCI5 per gram artificialdiet were used. A laboratory reared homogeneous culture of H.armigera was taken to carry out the bioassays. Three sets, each 30larvae at the early second instar stage were placed on artificial dietalone or artificial diet containingWCI2 orWCI5. The individual secondinstar larva was weighed and then placed in a container withappropriate diet. Larval diets were added or changed depending onconsumption. Larval weights were recorded every 48 h and larvaewere visually monitored for molting.

3. Results and discussion

3.1. Isolation of WCI genes and sequence analysis

Two putativewinged bean chymotrypsin inhibitors,WCI5 andWCI2were isolated by PCR approach. While WCI2 has been identifiedpreviously (Habu et al., 1992), WCI5 represents a novel putativeproteinase inhibitor (Table 1S). Both WCI2 and WCI5 have an openreading frame of 624 nucleotides that would encode proteins of 207

amino acids with a sequence similarity of 86% and a calculatedmolecular mass of 22.7 kDa. Since the first 24 amino acids constitute ofa signal sequence as previously reported for WCI3 (Peyachoknagulet al., 1989), the mature inhibitors probably consist of 183 amino acidresidues (Fig.1). LikeWCI2 and other knownKunitz-type chymotrypsininhibitors, the WCI5 polypeptide chain contains four half-cysteineresidues and the typical Leu65–Ser66 reactive site (Fig. 1, Habu et al.,1992; Kortt et al., 1990). Relatedness of the winged bean Kunitz-typechymotrypsin inhibitors was confirmed by phylogenetic analysis usingthe winged bean chymotrypsin inhibitors and Kunitz-type inhibitorproteins from other plant species (Fig. 1S). The inhibitor proteins fromwinged bean group together with Erythrina variegata chymotrypsininhibitorswhile the trypsin inhibitors from the other plant species formseparate groups. This analysis revealed total 5-major subgroups basedon the sequence similarity. Earlier reported WCI2 and WCI3 showedhigh similarity (Peyachoknagul et al., 1989) while WCI5 exhibit highsimilarity with WCIx.

Fig. 1. Analysis of WCI2, WCI5 and related protein sequences. (A) Amino acid sequencesof WCI2 and WCI5 and alignment with two related members of the CI family in wingedbean (Table 1S). The sequence alignment was generated by the Clustal algorithm. Blackbackground shows identical and similar amino acids. The highly conserved singlepolypeptide chain with four half-cysteine residues that form two intracellulardisulphide bridges (⁎) and the Leu65–Ser66 reactive site (boxed) are highlighted.

Fig. 2. Organ-specific expression of WCI2 and WCI5 in winged bean plant. Semi-quantitative RT-PCR was carried out using WCI gene-specific primers and total RNAfrom different plant organs. For control of even amounts of RNA in each tissue sample,18S rRNA was amplified. Seeds include seed coats and cotyledons; seed I, 40 days afterflowering (DAF); seed II, 90 DAF.

Fig. 3. Analysis of WCI recombinant proteins. (A) SDS-PAGE with purified proteinfractions of winged bean PIs expressed in P. pastoris. WCI5 (lane 1), WCI2 (lane 2) andempty vector (lane 3) alongwith a proteinmolecularweight standard (M). (B) SDS-PAGEfollowed by activity visualization with chymotrypsin and gel-X-ray film contact printmethod. Protein of WCI5 (lane 1), WCI2 (lane 2), empty vector (lane 3) expressing P.pastoris cells and seed extract ofwinged bean (lane 4)were separated on 12% SDS gel andvisualized by the X-ray film contact print method (Pichare and Kachole, 1994) for theiractivity against chymotrypsin (0.05%). Winged bean seed extract containing highamounts of proteinase inhibitor proteins was used as positive control.

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3.2. WCI2 and WCI5 are expressed in winged bean seeds

To analyze if WCI2 and WCI5 expression patterns in winged beanare consistent with their predicted function in insect defense inseeds, tissue-specific expression studies were performed by RT-PCR(Fig. 2). Analysis of the WCI2 transcripts demonstrated strongexpression in winged bean seeds, which is consistent with thepreviously published WCI2 expression data (Habu et al., 1992). Incontrast, low levels of WCI2 transcripts were also found in leaves,flowers and pod-walls (Fig. 2). These differences could be attributedto the sensitivity of the methodology used as low levels of transcriptscan be detected by RT-PCR.WCI5 expressionwas exclusively observedin developing and mature seeds (Fig. 2). High expression level ofWCI2 and WCI5 in developing and mature seeds of winged beanshown in this study (Fig. 3) is consistent with the proposed role ofseed PIs in defense against H. armigera.

3.3. Analysis of recombinant inhibitor proteins

WCI2 and WCI5 were expressed in yeast cells and purified proteinwas analyzed by SDS-PAGE. Protein extracts showed each a singleband of "20 kDa, similar to the predicted size of WCI2 and WCI5,respectively (Fig. 3A). Analysis of expressedWCI2 andWCI5 protein byvisualization with chymotrypsin and X-ray film contact printtechnique showed single activity band for each protein extract,indicating that both proteinase inhibitors can be successfullyexpressed in yeast (Fig. 3B).

Besides WCI2, two other WCI genes/proteins have previously beenisolated from winged bean (WCI3 and WCIx, Table 1S), but only WCI3has been characterized for its biochemical properties and was shownto inhibit bovine " chymotrypsin at 1:2 ratio of inhibitor:enzyme(Kortt, 1980; Kortt et al., 1990). Commercially available bovinechymotrypsin and trypsin (here onwards called chymotrypsin andtrypsin) as well as different HGPs were tested for inhibitory activity ofWCI2 and WCI5 proteins by enzymatic assays. Chymotrypsin (2.4 !g)was first titrated against various concentrations of WCI2 and WCI5 toobtain maximum possible inhibition. At low protein concentrations(0.3 !g and 0.6 !g)WCI2 demonstrated a stronger inhibitory effect thanWCI5. Chymotrypsin activity was inhibited by 22% and 46% withWCI2whileWCI5 had an inhibitory effect of 9% and 25%, respectively. At highconcentration (2.4 !g), WCI5 inhibited chymotrypsin activity com-pletely while inhibition by WCI2 was slightly less efficient with 92%.Like WCI3, WCI2 did not completely inhibit chymotrypsin activity,whichwas expected asWCI2 differs fromWCI3 by only one amino acid.In contrast,WCI5 inhibited chymotrypsin to 100%. Interestingly, at lowPI concentrations WCI5 inhibition of chymotrypsin activity was weakcompared to WCI2 (Fig. 4A).

WCI2 and WCI5 (3.8 !g each) inhibited trypsin activity to amaximum of 28% and 63%, respectively (Fig. 4B), while WCI3 analyzedin earlier studies did not inhibit trypsin (Ghosh and Singh, 1997;Shibata et al., 1986; Kortt, 1980; Khamrui et al., 2005). Substitution ofHis (137) in WCI3 to Arg (137) in WCI2 and WCI5 (Fig. 1; 24) might beresponsible for trypsin binding and inhibition since the arginineresidue is a cleavage site for trypsin. Although the majority of theproteinase-PI contacts are probably made by the P1 residue of theinhibitor (Khamrui et al., 2005), extended binding interactionsbetween the proteinase and the PI could contribute to the stability

Fig. 4. Chymotrypsin and trypsin inhibition by WCIs. (A) Percent inhibition ofazocaseinolytic activity of chymotrypsin by WCI2 and WCI5. 0.4 U of azocaseinolyticactivity of chymotrypsin (2.4 !g) was titrated against increasing concentrations ofWCI2and WCI5. Linear segment of the curve was extrapolated to estimate the molar ratio ofinhibition. WCI2 and WCI5 inhibited up to 92% and 100% of the chymotrypsin activity,respectively. Each value is an average of three repetitions and standard errors isindicated by error bars. (B) Percent inhibition of azocaseinolytic activity of trypsin byWCI2 and WCI5. 0.4 U of azocaseinolytic activity of trypsin (1.2 !g) was titrated withincreasing concentrations of WCI2 and WCI5 and inhibition curve was plotted. WCI2andWCI5 inhibited up to 28% and 63% of the trypsin activity, respectively. Each value isan average of three repetitions and standard errors is indicated by error bars.

Fig. 5. Inhibition of the gut proteinases of H. armigera (HGP) by WCIs. (A) Inhibition ofHGPs of H. armigera larvae fed with artificial diet (cHGP). 0.4 U of azocaseinolyticactivity of cHGP (1.4 !l) was titrated with increasing concentration of WCI2 and WCI5and inhibition curve was plotted. Each value is an average of three repetitions andstandard error is indicated by error bar. (B) Inhibition of HGPs of H. armigera larvae fedwith host plants or PI-containing diets. HGPs were obtained from larvae fed on okra(ok), pigeonpea (pp), chickpea (cp) and larvae grown on artificial diets supplementedwith Momordica charantia trypsin inhibitor-II (mc), winged bean seed PIs (wb) andpotato inhibitor-II (PIN-II). Inhibition of various HGPs by WCI2 and WCI5 (3.5 !g each)was analyzed by azocaseinolytic activity assay. Each value is an average of threerepetitions and standard errors are indicated by error bars.

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of the association (Srinivasan et al., 2006). In case of a leech bacterialsubtilisin inhibitor it has recently been demonstrated that changes in‘adventitious contacts’ are responsible for the changes in affinity forthe target enzyme or discrimination of an inhibitor for closely relatedtarget proteinases (Komiyama et al., 2003). Determination of thecrystal structure of both WCI2 and WCI5 and comparison with theWCI3 structure might help to explain the variation in chymotrypsinand trypsin inhibition by these proteins.

WCIs were also titrated against the gut proteinases of fifth instar H.armigera larvae grown on artificial diet (cHGP). Compared to WCI2,WCI5 showed stronger inhibition of cHGP activity at all inhibitorconcentrations tested. Maximum inhibition of cHGP activity was 56%and 78% with 3.5 !g of WCI2 and WCI5, respectively (Fig. 5A). Infurther experiments, both the inhibitors (3.5 !g) were used to analyzeinhibition of gut proteinases from H. armigera larvae fed on variousdiets. When fed on plants such as okra (ok), pigeon pea (pp) andchickpea (cp) inhibition of HGPs by WCI2 was between 32 and 41%,while inhibitory effects of WCI5 were up to 58% (Fig. 5B). WCI2 andWCI5 both exhibited stronger HGP inhibition when the larvae werefed with artificial diet in the lab compared to those fed with okra,pigeonpea, and chickpea in the field (Fig. 5). Stronger inhibitory effectof PIs on HGP from a homogeneous laboratory-grown insect culturehas been observed earlier (unpublished data). A slight increase inWCI2 and WCI5 inhibition of HGPs was n when larvae were grown onartificial diets supplemented with PIs from M. charantia (McTI-II),winged bean (wb) and potato (PIN-II). Inhibitory activity of WCI2 andWCI5 were as high as 58% and 69%, respectively (Fig. 5B). Inhibition ofHGP activities from larvae fed on PI-incorporated diets was similar tocHGP inhibition indicating that WCIs could effectively inhibit activityof PI-induced HGPs. Compared to WCI2, WCI5 generally seemed to bea stronger inhibitor of HGPs as well as of commercial trypsin andchymotrypsin.

3.4. Anti-metabolic effects of recombinant WCIs on growth ofH. armigera larvae

To analyze if WCIs affect the growth and development of H.armigera larvae, feeding studies were carried out using artificial dietwithout/with WCI proteins. At each instar, larvae fed on control diet(no PI) weighed significantly more than those fed on inhibitor-incorporated diets. By the end of second instar (day 2)WCI2-fed larvaedemonstrated a 20% reduction in larval weight which went up to 49%at the fifth instar (day 14) compared to control larvae. Similarly,

ingestion of WCI5 led to a 64% and 56% reduction in larval weight bythe end of second and fifth instar, respectively (Fig. 6). At the fourthand fifth larval instar, WCI5-fed larvae weighed less than larvae fedwith WCI2 diet indicating consistent and stronger inhibitory activityof WCI5 compared to WCI2. The concentration of WCIs (20 !g/g ofartificial diet) used in the present assay is much lower as compared tosome other Kunitz-family inhibitors such as SKTI (0.1–2.0% w/w) andCaKPI (40.2 !g/g of artificial diet) used to demonstrate their anti-metabolic effects on H. armigera (Johnston et al., 1993; Bown et al.,1997; Srinivasan et al., 2005). In earlier PI feeding studies, Marchettiet al. (2000) observed that PI-fed larvae gradually lost their turgor andbecame shrunken; apparently food avoidance had a dramatic effect onthewater balance of the larvae. The presence of PIs in the diet reducedflow of food in the midgut of larvae, which led to food refusal. Wenoted similar changes in the PI fed larvae during the course ofbioassay. Due to the altered feeding behavior, the larvae grewirregularly on inhibitor incorporated diets. Larval stage of H. armigerabeing the only actively feeding stage in the life cycle, female insectsaccumulate large reserves of proteins necessary for egg-laying duringthis period. Lower average body mass of larvae could thus translate into reduced fecundity of female moths (Telang et al., 2000; 2002). Onepossible reason for high efficacy of WCIs to H. armigera is due toselection of inhibitors on the basis of inhibitory potential againsttarget insect gut proteinases.

Severalplant PIshave alreadybeen transferred into insecthost plantslike tobacco and tomato to test their potential in defense against variousinsect species (De Leo et al., 1998, 2001; Charity et al., 1999; for reviewsee Jouanin et al.,1998). However, insect feeding studies on these plantshave shown confounding results. Overexpression of a PI from Alocasiamacrorrhiza (giant taro) in tobacco plants resulted in growth anddevelopmental retardation in H. armigera (Wu et al., 1997). Transgenictobacco and pea plants expressing a multi-domain PI from Nicotianaalata exhibited increased mortality and delayed growth in H. armigeralarvae feeding on the leaves (Charity et al., 1999). On the contrary, SBTIfrom soybean (Glycine max), in spite of its high expression in transgenictobacco plants, failed to confer resistance against H. armigera (Nandi etal., 1999). AsWCIs in the present study have been isolated from a ‘non-host’ plant, it would be interesting to study their insect inhibitory effectsby transgenic expression in a suitable host plant. Our hypothesis forselecting non-host plants for screening potential inhibitors of H.armigera proteinases was based on that insect is not exposed to theseinhibitor(s). The differences in inhibitor sequences exhibit in the non-host plants compared host-plant inhibitor sequences might contributefor high activity against H. armigera proteinases. We are in process ofestablishing the structure-functional correlation by studying insectproteinase–proteinase inhibitor (host and non-host plant PIs) in silico aswell aswith co-crystallization studies. Such studiesmight providemoreinsight in to these interactions and explain contribution of few sequencevariations in the activity.

3.5. Conclusions

Two putative Kunitz-type chymotrypsin inhibitor genes (WCI2 andWCI5) were isolated from winged bean (P. tetragonolobus (L.) DC) andexpressed as recombinant proteins using P. pastoris. Both theinhibitors strongly inhibited the gut proteinases and growth of H.armigera. The inhibitory effects of WCI2 and WCI5 on activity ofproteinases and larval growth make these proteins and their genespromising candidates for enhancing plant defense against H. armigerausing transgenic plants.

Acknowledgments

We acknowledge the technical support of Nathan Tarlyn and theassistance of Chuck Cody in growing the plants at Washington StateUniversity (WSU). This work was supported by the McKnight

Fig. 6. Effect of WCI ingestion on growth of H. armigera larvae. Recombinant WCI2 orWCI5 proteins were employed in controlled feeding experiments. H. armigera larvaewere allowed to feed on artificial diet without any inhibitor (control) or with WCI2 andWCI5 incorporated diets (20 !g inhibitor protein/g diet). Larval weights were recordedevery alternate day beginning after two days of feeding. Developmental stages of larvaeduring feeding period are II, second; III, third; IV, fourth and V, fifth instar. Graph showsaverage weights of minimum 30 larvae for each feeding set.

84 M.A. Telang et al. / Gene 431 (2009) 80–85

Foundation International Collaborative Crop Research Program grant# 99348 to WSU and GAP011526 to NCL. MAT was supported by aSenior Research Fellowship from the Council of Scientific andIndustrial Research, Government of India, New Delhi.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.gene.2008.10.026.

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