plant insecticidal proteins and their potential for...

Indian Journal of BiotechnologyVol 2, January 2003, pp. 110-120

Plant Insecticidal Proteins and their Potential for DevelopingTransgenics Resistant to Insect Pests

K R Koundal* and P RajendranNational Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India

Insects cause heavy damage to cultivated crop plants. Production of proteinaceous inhibitors that interfere withthe digestive biochemistry of insect pests is one of the naturally occurring defence mechanisms in plants. These pro-teins include lectins, arcelins and inhibitors of alpha amylases and proteases of various larvae pests. Use of plantgenes encoding effective inhibitors of major digestive enzymes, such as protease and a-amylase inhibitors of the tar-get pest species is emerging as viable approach for the production of pest resistant transgenic crop plants. Therefore,it is important to characterize proteins and their genes from our indigenous crops in order to strengthen andbroaden our gene bank for pest control manipulations. The availability of diverse insecticidal proteins and theirgenes from different plant species will make it easier to use one or more genes in combination to develop resistantcrop plants. Eventually, insect resistant transgenic plants will certainly prove more economic than any conventionalcontrol strategy if long-term benefit of transgenic crops especially factors such as environmental damage and humanhealth risks are considered.

Introduction

Keywords: plant proteins/genes, protease inhibitor, lectin, a-amylase inhibitor, transgenics, bioassays

Pest infestation leading to massive crop damage isthe most serious limiting factor in crop productivity.In the course of evolution, many classes of proteinsand secondary metabolic substances have been ex-pressed in plants as effective counter measures againstherbivory and the application of these natural plantdefence traits as candidates for genetic engineeringfor crop pest control is now a reality. Plant secondarymetabolites include a plethora of biochemical entitiesthat are synthesized through complex pathways andtherefore, the attempts to explore their biochemistryand physiology have always remained challenging.Broadly, they belong to totally unrelated classes ofcompounds like alkaloids, f1avonoids, foliar enzymes(polyphenol oxidases & peroxidases), foliar phenolicacid esters (rutin & chlorogenic acid), non-proteinamino acids, peptide hormones, pyrethrins, steroids,

"Author for correspondence:Tel: 91-011-25788783, 25711554Fax: 91-011-25823984,25766420E-mail: [email protected]:Bt: Bacillus thuriengiensis; a-AI: Alpha-amylase; Pi: Proteaseinhibitor; CpTi: Cowpea trypsin inhibitor; Ti: Trypsin inhibitor;Cti: Chymotrypsin inhibitor; GNA: Glanthus nivalis; cDNA:Complementary deoxyribonucleic acid; CaMV: Cauli mosaicvirus; GM: Genetically modified.

and terpenoids. Yet another group, but more impor-tant from the application point of view belongs toplant defence proteins that are more specific againstinsect pests (Maqbool et at, 2001). In most plants,they offer horizontal resistance against insect attack(Fabrick et al, 2002). This review is exclusively fo-cussed on the potential of these inhibitors or their en-coding genes in pest control manipulations. Attemptsare made to depict the structural functional relation-ships of these inhibitors that direct their application todevelop transgenics resistants to various insect pests.

Plant Proteins with Insecticidal ActivityProduction of proteinaceous inhibitors that inter-

fere with the digestive biochemistry of insect pests isone of the naturally occurring defence mechanisms inplants. This mechanism is manifested in the form ofaccumulation of one or several defence proteins suchas lectins, arcelins and inhibitors of alpha-amy lasesand protease of larval pests. As insect larvae develop,they secrete into their guts a variety of enzymes thatbreak down carbohydrates and proteins present in theingested food. The larvae then feed on the releasedcomponents from the plant tissue rapidly causing cropdamage and yield loss. Plants respond to this invasionby synthesizing proteins that can inhibit the action ofthese enzymes. If a food source contains a supply ofdigestible protein or carbohydrate, but also an inhibi-

KOUNDAL & RAJENDRAN: PLANT INSECTICIDAL PROTEINS FOR TRANSGENICS RESISTANT TO PESTS 1 I I

tor that acts as a biological check, the larval enzymescannot digest the food and the larva will not grow anddevelop through its normal cycle (Boulter, 1993; Fab-rick et al, 2002).

The potential for using this natural host plant re-sistance in pest control across the plant genetic barri-ers has increased with the development of gene trans-fer techniques. Though exogenous natural pesticidalagents such as Bt toxin are also effective in deterringplant predators, in spite of their current successes,they may create environmental safety and consumerhealth debates in future in addition to a multitude ofethical concerns (Gatehouse et al, 1998). Further,there is growing evidence that genes derived fromplants could be excellent alternatives to the toxinsfrom Bacillus thuringiensis. There are now severalexamples of protease inhibitors of plant origin confer-ring insect resistance when expressed in transgenicplants including tobacco, rice, cotton, strawberry,poplar and peas (Ussuf et al, 2001). Plant derivedgenes target different sites in insects than the syn-thetic chemicals and may be deployed in combinationwith the bacterial insecticidal genes for complete in-sect control. However, this approach can be exploitedeffectively if encoding genes for proteins with appre-ciable inhibitory properties on the target pest's en-zymes are available for genetic manipulations. Thus,it is imperative to evaluate as many plant sources aspossible for identifying the presence of proteins withideal insecticidal properties (Sparvoli et 01, 2001). Itis equally important to characterize these proteins andtheir encoding genes to strengthen and broaden theresistance gene pool. Among them, alpha-amylaseinhibitors, protease inhibitors and lectins have under-gone extensive investigations particularly in the lasttwo decades and the research on these proteins andtheir encoding genes has proved to be rewarding. Inaddition, some related but less studied proteins likearcelins and vicilins have also invoked research atten-tion in recent years.

(i) Alpha-amylase InhibitorsAlpha-amylase a ubiquitous enzyme, catalyses the

endolytic cleavage of 1~4 linked glucose polymers,mainly starch to give hydrolytic products with [he a-configuration. This digestive enzyme has been of in-terest to plant biologists for many years because of itsagricultural and industrial importance. In plants, a-amylase inhibitors are stored in seeds and tubers to-wards the end of the life cycle in copious amounts.

They are also synthesized in plants almost at the sametime and usually in the same organs that store thestarch and amylases. Their role is mainly defencethrough inhibition of a variety of alpha-amylases fromdiverse sources by forming irreversible complexeswith them. Chrzaszez & Janicki (1934) were the firstto report the presence of a-Als in buckwheat. There-after, a-AI has been isolated from many plants,mainly cereals and legumes. a-AI inhibitors are pri-marily the products of single gene, and are small, lowmolecular weight (in the 14-8 kDa range) monomericproteins, a-Als from bean contain two or more sub-units (Ho & Whitaker, 1993) and a-Als in wheat,barley and rye are tetrameric in nature (Garcia et al,1996; Sanchez-Monge et 01, 1996). Several a-AIproteins that have been isolated from wheat kernelsstrongly inhibit alpha-amylases from insects with nullor weak effect on mammalian amylases (Feng et 01,1996). These properties make them attractive candi-dates for plant genetic manipulations for impartingresistance to insect pests. Incorporation of increasingamounts of a-AI into the diet of the pea weeviliBruchus pisorum) was directly correlated with de-layed larval development time (Ishimoto & Kitamuraet al; 1992). The resistance of common beantPhaseolus vulgaris) seeds to bruchids like cowpeaweevil and adzuki bean weevil has largely been at-tributed to the presence of a-Als (Huesing et al,1991). The level of a-Als in their seeds has beenfound sufficient to inhibit the development of the lar-vae of those bruchid species that normally do not feedon common bean.

Though the cereal and leguminous a-amylase in-hibitors share a similar mode of action, studies ontheir structure and evolutionary relationships haveclearly shown that they belong to entirely differentclasses of proteins that might have evolved independ-ently in monocotyledon and dicotyledon in a conver-gent manner. The cereal trypsin/ a-AI proteins aremembers of a super family of seed proteins with lim-ited sequence homology. Homologous relationshipsof the cereal o-Als were established with the 2S stor-age proteins from castor bean and the Kazal secretarytrypsin inhibitor from bovine pancreas (Odani et 01,1983), thus showing that this protein super family isdistributed beyond the plant kingdom. The C_N-1._pr~-_teins from wheat, barley and rye were eventuallyfound to be members of the trypsin/a-AI family(Shewry et 01, 1984) and the suBtilisin/endogenous

112 INDIAN J BlOTECHNOL, JANUARY 2003

a-Als from cereals were found to be homologous tothe soybean trypsin inhibitor (Maeda, 1986).

On the other hand, the common bean a-amylaseinhibitor belongs to a family of vacuolar glycopro-teins that comprises phytohemglutinins (PHA), arce-lins and a-Als (Moreno & Chrispeels, 1989; Chris-peels & Raikhel, 1991). PHA is the true lectinwhereas arcelins may retain a weak carbohydratebinding activity and a-Als is completely devoid ofsuch activity.Their genes are tightly linked, behave asa single Mendelian locus, and have likely arisen byduplication and divergence of an ancestral gene (No-dari et al, 1993).Three dimensional models of a-AIs,arcelins and lectins have shown that arcelins and a-AIs are truncated lectins (Rouge et al, 1993). In thecourse of evolution, the loss of one or two loops lo-cated in the region of monosaccharide binding site inlectin led to the development of arcelins and a-IUs,respectively. This might be the reason why arcelinsexhibit a weak agglutinating activity, while a-Alscannot agglutinate erythrocytes. Further, an a-AI-likeprotein, YBP 22, isolated from the tuberous roots ofthe Mexican yam bean [Pachyrrhizus erosus (Linn.)Urban] showed sequence homology with severalknown protease inhibitors, and a polyclonal antibodyraised against the protein cross-reacted with soybeantrypsin inhibitor (Gomes et al, 1997). The cDNAclones of lectin related seed proteins isolated fromLima bean showed 93.7% nucleotide identity and en-coded an arcelin-like and an a-AI-like protein respec-tively (Sparvoli et al, 1998).

(ii) Protease Inhibitors (PIS)Read and Haas (1938) reported the presence par-

ticularly in legume seeds of proteases and their bio-chemistry as protein molecules was confirmed by Ku-nitz who isolated and crystallized the trypsin inhibitorfrom soybean (Kunitz, 1945). Protease inhibitors isthe largest class of proteins that have undergone ex-tensive investigations and consequently their struc-ture, properties, function and metabolism have beenwell documented. They are almost ubiquitous inplants and those present in the Leguminosae, Grami-nae and Solanaceae have been studied and character-ized in greater detail (Richardson, 1977; Connors etal, 2002). Although, some of them may playa role inendogenous protein metabolism, most of the proteaseinhibitors that have been characterized from plants donot inhibit endogenous plant proteases, but havespecificities for animal or microbial enzymes (Las-

kowski & Kato, 1980; Laskowski et al, 1988).111 vitrofeeding trials using artificial diets containing the in-hibitors have confirmed the protective role for prote-ase inhibitors against several crop pests. The effectsof Pis on susceptible insects are generally seen as anincrease in mortality, decrease in growth rate andprolongation of developmental period of the larvae.These detrimental effects are accomplished byblocking insect midgut proteinases thus impairingprotein digestion, which inhibits or at least delays (inthe case of weak inhibitors) the release of peptidesand amino acids from dietary protein. The presence ofinhibitor thus leads to the loss of nutrients particularlysulphur containing amino acids, and thereby weak and

·stunted growth and ultimate death (Gatehouse et aI,1992).

A systematic classification of Pis is not viable dueto their diversity in terms of source, structure, specifi-cities and size. However, these are mainly grouped inthe four specificity groups, viz. serine, cysteine, met-allo and aspartic protease inhibitors (inhibiting serine,cysteine, metallo and asparty I proteases, respecti vely)(Garcia-Olmedo et at, 1987). Among them, the potatoinhibitor 1 and 11 families, the Bowman-Birk inhibi-tor (BB!) family, and the soybean trypsin inhibitor(Kunitz) family are very important due to their in-hibitory potential on gut enzymes of major crop pests.

(iii) Potato Inhibitor 1and 11Families

Among the Pis, the wound-inducible inhibitorsfrom potato and tomato represent a unique group withinsecticidal properties due to several interesting fea-tures of these proteins and their encoding genes. Theycomprise a non-homologous gene family in whichmembers have been identified mainly from the sola-naceous plants. Among them, potato inhibitor I and IIand tomato protease inhibitor I and II have been wellcharacterized (Plunkett et al, 1982). The unique andmost striking feature of their encoding genes is thepresence of introns, two each in inhibitor I genes andone in the gene encoding potato inhibitor II. In fact,they are the only protease inhibitor genes reported sofar to contain introns. In potato alone, a mixture of tenor more isoinhibitors of protease inhibitor I and atleast three forms of inhibitor II have been identified.In addition, homologues of the inhibitor have beenfound in some non solanaceous plants like alfalfa.broad bean, clover, cowpea, cucumber, French bean.grape, squash, strawberry, barley and buckwheat(Lorene et al, 2001; Ye et al, 2001). In leaves of to-

KOUNDAL & RAJENDRAN: PLANT INSECTICIDAL PROTEINS FOR TRANSGENICS RESISTANT TO PESTS 113

mato and potato, they are expressed constitutively atlow levels during plant growth and development. Inresponse to wounding by insects or other mechanicaldamage, their concentration increases dramaticallyeven in the unwounded leaves of the same plant, andwithin a few hours of injury, their levels often exceed10% of total soluble proteins. In potato tubers, theyaccumulate throughout the course of tuber develop-ment, and represent a substantial fraction of the solu-ble protein. Thus, unlike other plant protease inhibitorgenes, these genes are regulated environmentally aswell as developmentally, and their expression is be-lieved to be under a complex control involving sev-eral cis and transacting factors making them excellentmodels for study of plant gene regulation (Keil et al,1986; Kouzuma et al, 2001).

All these proteins belong to the serine protease(endopeptidases) inhibitor group and are relativelysmall in size. Potato inhibitor I is a pentameric proteinof Mr 41000 and is made up of monomers of Mr 8100with single reactive site to inhibit chymotrypsin whereas the corresponding inhibitor species in tomato,which is also a pentamer, with subunits of Mr 7800each. Their cysteine content is low and they eitherhave one disulfide bridge or none and the presence ofcysteine seems to be not essential for their activity. Inother inhibitor families, disulfide bridges stabilize thethree dimensional structure and are believed to be es-sential for their activity. In the inhibitor II family,both in tomato and potato, the mature protein is a di-mer of about Mr 25000 made up of two promoters ofidentical size, with five disulfide bonds and two reac-tive sites per monomer, which inhibit chymotrypsinand trypsin respectively. Wound induction of theseinhibitor proteins is triggered by a small (18-mer)peptide hormone, systernin or Protease Inhibitor In-ducing Factor (PUP), released into the vascular sys-tem in response to wounding which in turn is trans-ported rapidly to other tissues of the plant where itinitiates the transcription of the PI genes. In addition,various factors including fungal elicitors, bacterialinfection, sucrose, abscisic acid, and jasmonic acidhave direct or indirect role in the increased synthesisof these inhibitors in potato. Nuclear runoff assayshave shown that the accumulation of inhibitors I andII in tomato is transcriptionally regulated (Graham etal, 1986). Once synthesized, the inhibitor I and Uproteins are sequestered into the central vacuole,which ensures their way long half lives. Analysis ofthe genomic clone encoding the potato protease in-

hibitor II has revealed the presence of sequencescharacteristic of typical plant signal peptides locatedimmediately downstream of the only intron present inthe gene (Keil et al, 1986). In fact, signal peptides ofmolecular weight around 2000-3000 daltons havebeen detected in newly translated inhibitor proteins.Moreover, simulation of the inhibitor induction waspossible in transgenic tomatoes carrying the prosys-ternin gene.

(iv) Bowman-Birk Inhibitor (BBI) FamilyThe trypsin subclass of serine protease inhibitors

from legume seeds exhibit insecticidal effects againstseveral crop pests belonging to the orders of Lepi-doptera, Coleoptera and Orthoptera (Shukle & Mur-dock, 1983). Many of these inhibitors are products ofmultigene families with varying specificities towardsdifferent proteases (Ryan, 1990). These inhibitors arecysteine-rich with a molecular mass of 8-20 kDa(Chen et ai, 1997).

The Bowman-Birk inhibitor (BB!) and its relatedfamily of isoinhibitors comprise a closely relatedgroup of serine Pis. The protein was first identifiedand isolated from soybean seeds by Bowman (1946)and further characterized by Birk and associates (Birket al, 1963), hence the name Bowman-Birk inhibitor(BBI). Later on, this class of inhibitors was isolatedfrom a large number of legumes and several otherplants including monocot. They contain higheramounts of sulphur amino acids and have a molecularmass less than 10,000 kDa. These proteins are classi-fied as double-headed serine protease inhibitors due tothe presence of two reactive site domains within thesame polypeptide, one each for trypsin (Lys-Ser) andchymotrypsin (Leu-Ser) molecules. These are productsof small gene analogues without introns (Hilder et al.1987; Boulter et al, 1989). The BBIs from seeds aredouble headed proteins of 8kDa, whereas the 8kDainhibitors from monocotyledonous seeds are singleheaded. However, 16 kDa double-headed inhibitorshave also been reported to be present in them. Com-parison of the primary structure of the 8 kDa the BBIdomains have revealed that a crucial disulfide whichconnects the amino and carboxy termini of the activesite loop, while present in the double-headed inhibitorsfrom dicotyledons is lost monocotyledons leading tothe loss of a reactive site and making them single-headed. Thus, the gene duplication event leading to a16-kDa double-headed inhibitor in monocotyledonsmight have occurred, probably after the divergence of

114 INDIAN J BIOTECHNOL, JANUARY 2003

monocotyledons and dicotyledons and also after theloss of a second reactive site in monocotyledons.

The cowpea trypsin inhibitor constitutes a some-what larger gene family of four major isoinhibitors,although the exact number of active genes is notknown. Three of the isoinhibitors are specific fortrypsin at each active site and fourth is a trypsin chy-motrypsin bifunctional inhibitor. Feeding trials onartificial diets have shown that the CpTI had inhibi-tory effects against a wide range of insects includingLepidopteran like Heliothis spp. (tobacco budwormand corn silkworm), Spodoptera (armyworms), Man-duca sexta, Coleoptera (beetles) including Calloso-brucus maculatus, Diabrotica sp. (corn rootworms),Anthonomous grandis (cotton boll weevil) and a rangeof other insects. This broad range is typical of manyplant based protection mechanism (Park et al, 2001).The trypsin inhibitor extracted from cowpea was amore effective anti-metabolite than those extractedfrom a number of other legumes (Boulter, 1993).Comparison of the content of anti-metabolites incowpea varieties has revealed that the seeds of thewild cowpea (Vigna vexillata) contained higheramounts of trypsin inhibitor and chymotrypsin in-hibitor and less of lectins compared to the cultivatedaccessions (Marconi et al, 1993). The high resistanceto bruchid and the high TI and CTI contents ofV.vexillata suggested that though there may not beany direct correlation between bruchid resistance andTI content, the protease inhibitors promote or at leaststrongly influence the plant's defence system.

The cowpea protease inhibitor protein is comprisedof readily indentifiable core region covering the in-variant cysteine residues and active serine centres thatare bound to highly variable amino and carboxy ter-minal regions. The specificity and binding propertiesof CpTI have been documented as attributes related tothe first and second domains comprising the core re-gion of the inhibitor protein. It is likely that isoformswith striking variations in inhibition and bindingproperties may be products of crucial mutations inregions adjoining the binding sites in one or both thedomains (Laskowski et ai, 1988). An added advantagefrom the biosafety point of view is that the cowpeatrypsin inhibitor is very sensitive to pepsin digestion,which is unlikely with many trypsin inhibitors. Sincethe ingested food is initially exposed to pepsin in themammalian digestive system, the CpTI may be com-pletely digested by pepsin rendering it safe for con-sumption.

The PI genomic clone (pLK8) isolated recently inour laboratory is from a genomic DNA library ofcowpea, cv 130 possessed an ORF which on transla-tion gave a protein with 167 amino acids (Lawrence etal, 2001). The molecular weight of this protein wascalculated to be 18.5 kDa; in which 18 amino acids,strongly basic, 20 strongly acidic, 40 hydrophobic and62 polar, with an isoelectric point of 6.5. The proteinhad a N terminal signal sequence of 69 amino acidswhich is important in its translocation as has beenreported in many proteinase inhibitors such as soy-bean (Hammond et al, 1984), cowpea, (Hilder et al,1989) pea, (Domoney et al, 1995), maize (Rohrmeiret al 1993), and alfalfa (McGurl et al, 1995). Thecowpea protease inhibitor had the signal peptidecleavage site between 69-70 amino acid residueswhich is comparable to the alfalfa trypsin inhibitor(ATI) having a signal peptide of 44 or 55 amino acidresidues which may target the ATI to the ER and theneventually deposit it in the vacuole of the cell(McGurl et al, 1995).

(v) LectinsLectins are defined as proteins possessing at least

one non-catalytic domain that reversibly binds to spe-cific carbohydrate(s). They are a heterogeneous groupof proteins differing from each other with respect totheir molecular structure, carbohydrate-binding speci-ficity and biological activities. In most plant species,they are found, at least in low amounts, in storage tis-sues of seeds, stems or bulbs (Esteban et al, 2002).There are four major sub-groups of plant lectins: thelegume lectins (arachis, soybean, lentil, kidney bean,pea, and faba bean), the monocotyledons mannosebinding lectins (onion, leek, taro & garlic), the chitinbinding lectins (barley, rice, wheat, rye, tomato &potato) and the type 2 ribosome inactivating proteins(castor bean & elderberry). However, heterologouslectins outside these groups have been reported frombanana, jackfruit and pumpkin. Among them, thosefrom leguminous plants form a large family of ho-mologous proteins with strong similarity at the levelof their amino acid sequences and tertiary structures,but with highly variable carbohydrate specificities andquaternary structures.

Over two decades ago, it was recognized that lee-tins possessed insecticidal properties. Lectins exerttheir inhibitory effect by binding to glycoproteins em-bedded within the peri trophic matrix lining the insectmidgut and in turn disrupting digestive processes and


nutrient assimilation. Ingestion of lectin results inboth binding of the lectin to membrane proteins in theinsect gut epithelium, and transport to the haemo-lymph, and may result in systemic effects on insectdevelopment. Wheat germ agglutinin was the mosteffective among 17 plant lectins screened for theirinhibitory effect on larval development of C. macu-latus (Murdock et al, 1990). While, the E-type lectins(erythrocyte-binding type) from common bean re-tarded the larval growth of C. maculatus (Gatehouseet al, 1984), the lectin from kidney bean did not thor-oughly inhibit the larval growth of the pest (Ishimotoet al, 1996) ..

The mannose-specific lectin (GNA) derived fromthe snowdrop, Galanthus nivalis, has been found to betoxic to major sap-feeding insect pests of agronomi-cally important crops like rice (against Nilaparvatalugens & Nephotettix virescens), and pea (againstCallosobruchus maculatusi, and has been expressedin at least nine food crops including oil seed rape,potato, rice, and tomato (Sauvion et al, 1996; Gate-house et al, 1996; Zhang et al, 2000). The range oftoxicity of GNA has recently been extended to theLepidopteran, Lacanobia oleracea (tomato moth),where it was observed that the mean larval biomasswas reduced by approximately 50% when reared upontransgenic potato (Fitches et al, 1997). In transgenictobacco expressing the pea lectin (P-lec) gene, the leafdamage caused by the larvae of Heliothis virescenswas greatly reduced (Boulter, 1990; Ye & Ng, 2001).The snowdrop lectin gene has also been successfullyexpressed in potato to give protection against aphids(Hilder et al, 1995), lepidopteron pests, and in riceagainst the rice brown plant hopper tNilaparvatalugensi.

Potential of Insecticidal Proteins for DevelopingTransgenics Resistant to Insect Pests

In general, transgenic plants developed using pro-tein inhibitors of insect digestive enzymes with a viewto control crop pests are designed not to kill the in-sects that feed, but to retard their development. Andpresumably, this is the fundamental difference be-tween this strategy and the chemical pest control oruse of Bt toxins that are aimed at complete controlthrough pest mortality. Thus, perceived effects of theinhibitors on a pest population are usually much lessdramatic than in the case with synthetic chemicalpesticides. Complete control of insects cannot be ex-pected in any realistic trial, tending rather to increase

mortality to a limited extent but to retard insectgrowth and development significantly. However, inan integrated pest management programme, cropprotection is accomplished through the concerted ef-fects of several complementing control measures.Moreover, the inhibitory effect of PIs could improvethe efficiency of defence proteins like Bt toxins or theplants' own defence proteins by preventing their deg-radation by the target pest proteases (Jongsma &Bolter, 1997). Therefore, even in situations wheretransgene expression does not keep the pest popula-tion below the threshold for intervention, it shouldallow a much wider window within which interven-tion can besuccessfully employed.

The first ever transgenic plants were produced byHilder et al (1987) using cowpea trypsin inhibitorcDNA clone. The transgenic plants were resistantagainst herbivorous insects such as collosobrchusmaculatus, Heliothis spodoptera and Diabrotica spand Tribolium sp. Johnson et al (1989) transformedtobacco plants with gene coding tomato and potatoinhibitor proteins and the transgenic plants found re-sistant to M. sexta.

Sane et al (1997) amplified the cowpea genomicDNA by polymerase chain reaction and cloned thefragment in a plant expression vector coupled withCaMV 35S promoter and NOS terminator and usedfor tobacco transformation. The transgenic tobaccoplants were tested against Spodoptera litura and werefound resistant. Recently, a protease inhibitor geneisolated from a native variety of cowpea in laboratorywas used to transform pigeon pea through Agrobacte-rium tumefaciens-mediated genetic transformation(Lawrence et al, 2001). The gene was driven byCaMV 35S promoter containing kanamycin resistanceas plant selection marker. Molecular analysis of the invitro cultured transgenic pigeon pea plants confirmedthe integration and stable expression of the proteaseinhibitor gene (Lawrence & Koundal, 2001). The firstgeneration of these transgenic pigeon pea plants isbeing maintained in glass houses for insect feedingexperiments and preliminary insect bioassays showedencouraging results against pod borer tHelicoverpaarmigera). Duan et al (1996) produced transgenicplant of rice by using wound inducible expression ofpotato proteinase inhibitor II. Bioassay of fifth gen-eration transgenic rice plants showed increased resis-tance to major rice pest, pink stem borer. Similarly,lectin genes have isolated and transformed into cropplants. Transgenic plants expressing snowdrop lectin


(Gatehouse et al, 1997), pea lectin (Boulter et al,1990) are found resistant to sap sucking insects. Re-cently, we have isolated and characterized a lectincDNA clone from cowpea (Datta et al, 2000). Thegene was introduced into Brassica using an expressioncassette. Comprising the CaMV S35 promoter, GUSand NOS terminator. Other moelcular analysis oftransgenics and in vitro feeding trails for testing theirefficacy against pea aphids showed significant in-hibitory effort on the growth and development (un-published). However, efforts are being made to isolateand characterize lectin from other legumes for identi-fying the novel genes that affect the insect pests buthave no effect on non-target organisms but present norisks in human health.

When ex-AI was expressed at high levels in trans-genic peas, weevil larval development was completelyblocked and the effects of weevil damage on overallseed yield was significantly reduced (Schroeder et al,1995). Larval development of several species of CaZ-losobruchus beetles, a pest of adzuki bean, was com-pletely inhibited when fed transgenic pea seeds ex-pressing the alpha-amylase inhibitor from commonbean. At the same time, Zabrotes subfasciatus, a natu-ral bruchid pest of common bean, could freely feed onthe transgenic plant (Ishimoto et al, 1996). Recently,an international collaboration between MaartenChrispeels, University of California and T J Higgins,CSIRO, Australia conducted the first field trials withtransgenic pea plants carrying ce-Al genes fromPliaseolus vulgaris. The genes encoding two ex-AIisoforms, alpha AI-l isolated from the cultivatedcommon bean (P. vulgarisi and alpha AI-2 gene froma wild accession, were introduced into pea (Pisumsativum) cultivars by using an Agrobacterium system(Morton et al, 2000). These transgenic plants ex-pressed high levels of ex-AIs in their seeds. Significantmortality of the first and second instar grubs wasfound with the ex-AI-l lines resulting in less weevilemergence than the control plants. Emergence wasdelayed in the ex-AI-2 lines but further analysisshowed that the exAI-2 had no effect on overall mor-tality. Initial safety studies on mammals showed that300 or 700 g of peas Kg-I of feed had no impact onthe nutritional value of transgenic peas fed to rats(Puzstai et al, 1999) have also demonstrated that theo-Als cause minimum mammalian toxicity in com-parison with the other plant inhibitor proteins. Thetoxic effects of alpha-amylase inhibitors vary with thespecies of insect amylase. The variation in sensitivity

towards the inhibitor may be attributed to either thepresence of inhibitor-insensitive amylases or to thesecretion of proteases that can degrade the inhibitor inthe larval midgut (Bown et al, 1997; Gatehouse et al,1997). The availability of such genes from differentplant species make it easy to use one or more genes incombination to develop transgenics for durable resis-tance against insect pests. The transgenic technologymay not replace the use of chemical pesticides in fu-ture but effectively complement it.

Constraints of Transgenic Crops

There are several constrains and apprehensions re-garding genetically modified GM food crops. Theseinclude: toxicity, allergenicity, carcinogenicity, use ofantibiotic resistant genes and nutritional value. Deci-sion regarding safety should be based on the nature ofthe product rather than other method by which it hasmodified. It is more important to bear in mind thatmany of the crop plants used today, contain naturaltoxins and allergens health hazards from food andhow to reduce them is an issue. Every effort should bemade to avoid the introduction of known allergensinto food crops and foods have to be clearly tabelledworldover. The concerns have been raised that thewidespread use of antibiotics resistance and genes asselective markers in plant could increase the antibioticresistance in human pathogens. Plant breeders shouldcontinue to remove all such markers from GM plantsand utilize alternative markers for the selection ofnew varieties.

Most of the environmental concerns about GMtechnology in plants have derived from the possibili-ties of gene flow to close relative!' of transgenic plantscreating super weeds or causing gene pollution amongother crops. There is a need for thorough risk assess-ment of likely causes of concern, at an early stage inthe development of transgenic plants varieties andevaluate these risks in subsequent field tests and re-leases. Internal agricultural community of every na-tion should support serious efforts to establish a sys-tem to adequately assess and also avoid human healthand environment risks. India is among a few countrieswhere regulation and guidelines on research andmonitoring of transgenic plants and their food safetyassessment have been are developed by the Depart-ment of Biotechnology, Govternment of India. How-ever, certain amount of risk is inherent in every newtechnology and a careful risk benefit analysis is nec-essary for making balance decision.


In spite of all the ideological dispute and ensuingdemocratic disagreements on the acceptability of ge-netically modified plants, genetic engineering for pestcontrol has now been realized as an environmentallybenign, flexible technology that can be safely fitted inany integrated pest management protocol. Further-more, it allows the possibility of developing entirelynew biological insecticides that retain the advantagesof classical biological control agents, but have fewerof their drawbacks. In reality, insect resistant trans-genic plants will certainly prove to be more economicthan any conventional control strategy if the long-term benefits of transgenic crops especially factorssuch as environmental damage and health risks areconsidered.

Future StrategiesThe use of insect resistant transgenic plants is a vi-

able means of producing crops with significantly en-hanced level of resistance. Several transgenic plantsexpressing plant-borne inhibitor proteins have beendeveloped in the last decade. Various approaches thatare being proposed and tried by different researchgroups include:

(i) Gene Combinations/Packaging/PyramidingThe protective efficacy, spectrum of activity and

the durability of resistance offered by the introducedgenes can be greatly enhanced through careful designof packages of different genes that contain compo-nents which would act on quite different target in-sects. Protease inhibitors may have a major role insuch gene pyramiding approaches. Apart from theirinherent insecticidal property, they would protectother introduced gene products from premature di-gestion in the insect gut and improve the overall per-formance through their mutually complementing orsynergistic effects. The first demonstration of this ap-proach has been the introduction of both cowpea tryp-sin inhibitor and pea lectin in transgenic tobaccoplants where the two gene products had an additiveeffect on tobacco bud worm caterpillars (Boulter et al,1990). It may be a useful approach to combine genesthat encode proteinase inhibitors among themselves oralong with suitable lectin, a-AI and/or Bt genes sothat multiple pest resistance may be achieved in a sin-gle event in agronomically important crop plants.Cross breeding of primary transformants carrying thedesirable gene combinations would also prove usefulin terms of enhanced insect resistance (Schuler et al,1998).

(ii) Protein EngineeringIn-depth exploration of protein structure and func-

tion may allow researchers to use protein engineeringas a strong tool for designing novel chimeric proteinsfor insect control. These chimeras are constructed bytailoring together the sequences that encode discretedomains of the protein intended to act on defined tar-gets. In vitro mutagenesis can be exploited for creat-ing very effective chimeric genes carrying desirabledomains with defined activity spectrum. The long-term goal of protein engineering would be the con-struction of modular protein that will target specificpests without any harmful effects on the beneficialorganisms. In principle, any domain from any proteincan be used in this modular system to construct pro-teins with a given set of attributes. Although still in itsinfancy, protein engineering will allow us to designproteins for use against the most insect pests.

(iii) Single-chain AntibodiesThis approach makes use of engineering antibodies

or antibody fragments specific to the target pest's es-sential protein and expressing it in the crop plant sothat both specificity and efficacy of action can be in-corporated in a single event (Hilder & Boulter, 1999).Besides, it has an additional advantage of avoidingaction on the non-target organisms, particularlypredators.

(iv) Phage DisplayThe technique combines in vitro ruutagemsis, ra-

pidity of molecular cloning, specificity of protein-protein interactions, and precision of molecularscreening techniques. After isolation and cloning ofan ideal inhibitor gene, a large collection of its vari-ants are prepared in the form of a library by alteringits sequence at every possible positions in the regionscritical for its action. In fact, the gene can be modifiedfor all the coding frames with every codon for each ofthe 20 possible amino acids so that the resultantchanges in specificity, binding and other attributes ineach of the modified product (protein) can be exam-ined. The cloned genes are then expressed on the sur-face of phage particles and the displayed proteins arescreened for the variant inhibitor protein, which ex-hibits the maximum affinity (binding) for the targetprotease enzyme. Thus, such technique envisages thescreening of millions of cloned proteins with the de-sired one being physically separated from othersbased upon its affinity to the target larval enzyme.


(v) Directed Protein EvolutionThis technique, a variant to phage display may turn

to be very useful in screening of large population ofinhibitor variants for their specificity with greater ac-curacy. Here, protein and peptide libraries are used toselect interactive proteins (ligands) without prior in-formation concerning their sequence or structure. Itcan be used to construct a phage display library ofmutant proteins or peptides as fusion proteins on thesurface of the phage. The library can then be used forscreening any enzyme for its binding specificity sothat the inhibitor with maximum affinity towards theenzyme can be easily located. The possible outcomeof the aforesaid developments would be the availabil-ity of the technology to overtake the insect's molecu-lar potential for resistance development through moreprogrammed, eco-friendly and efficient plant geneticengineering strategies.

ConclusionThe continuous use of pesticides for crop protection

had resulted in damaging impact on biological ecosys-tems, bio-magnification effects through pesticide resi-due deposition in food and feed pollution. The use oftarget specific compounds with low persistence and theexploitation of intrinsic plant resistance mechanismsare suggested as safer alternative strategies for effectiveinsect pests management. Thus, insect resistant GMplants will curtail the use of those hazardous pesticidesby engineering genes that encode natural biodegradableproteins with no harmful effect to animals and humanbeings. It is important to remember that biotechnologytools complement and extend the traditional methodsused to enhance agricultural productivity and to de-velop new production systems. The progress of agri-culture in developing, non-industrialized countrieswhere the economic activity still provides 60-80% em-ployment and 50% of national income will be possible.The application of plant biotechnology will be nodoubt, more meaningful in the poor and non-industralized parts of the world where socio-economicdevelopment will be possible only through sensibleadoption of advanced agricultural practices that helpfor enhanced food, feed' and fibre production and anoverall improvement in living standard. Despite, theopposition from the anti-GM movements all over theworld, undoubtedly, will find its more and immediateapplication in insect pest control of major crops. Theavailability of diverse insecticidal genes from differentplant species makes it a possibility to use one or moregenes in combination whose products are targetted at

different biochemical and physiological processes. Thetransgenic crops developed for insect resistance shouldbe compatible with other components of integrated pestmanagement programmes for pest resistance to be du-rable and effective. It is envisaged that it will have amajor impact on agricultural systems both in the devel-oped and developing countries in the near future.

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