Principle and ectopic expression of entomotoxicbiological molecules

Bt protein (Bacillus thuringiensis)

Bacillus thuringiensis (Bt), a well-known gram-positive bacterium,produces δ-endotoxins, or insecticidal crystal proteins (Cry proteins)during the sporulation phase [14]. The crystals, upon ingestion by theinsect larva, are get solubilized in the alkaline midgut into individualprotoxins. These protoxins are acted upon by midgut proteaseswhich cleave them into two halves, the N-terminal half is responsiblefor the production of the toxic Bt protein. This crystal protein wasexploited in the plant transgenic research to overcome crop damageby insect pests. The Belgian company Plant Genetic Systems was thefirst company to develop a genetically engineered plant with insecttolerance by expressing a cry gene from Bt in tobacco in 1987. Thetobacco plants engineered with cry1Aa genes and the cry1Ab toxinswere found to be resistant to the larvae of Manduca sexta (tobaccohornworm) [15]. Subsequently, many crop plants which includecotton, rice, maize, peanut, soybean, canola, tomato, potato andcabbage were transformed with various modified cry genes [16]. Thecommercialization of Bt-crops started in 1996 with the introductionof bollworm-resistant cotton (‘Bollgard’) in USA. Introduction ofBt endotoxin genes into crops became immensely popular, and it isestimated that Bt crops are cultivated in 66 m hectares area worldwide[17]. However, these transgenic crops expressing Bt endotoxin havebeen effective in controlling chewing pests but are less effective inproviding protection against sap-sucking pests [18]. Despite thegreat commercial successes of Bt crops, there are growing concernsabout the ability of insect to develop resistance to the Bt endotoxins[17,19, 20]. In contrast to Bt crops, alternative sources of potentialinsecticidal gene products need to be explored. To date, severaldifferent classes of proteins including lectins, ribosome-inactivatingproteins, protease inhibitors and α-amylase inhibitors and manymore have been shown to be insecticidal effects towards a range ofeconomically important insect pests by direct assay or by expressionin transgenic plants [21,22].

Vegetative Bt protein

A novel class of proteins called vegetative insecticidal proteins(Vips) produced by Bt during its vegetative stages of growth have beenidentified [23]. Although B. thuringiensis δ-endotoxins are effectiveinsecticidal proteins, there are several agronomically importantinsects that are less sensitive to their action. The 88 kDa vegetativeinsecticidal protein has a putative bacillar secretory signal at theN-terminal which is not processed during its secretion. Vegetativeinsecticidal proteins (Vip) are effective against lepidopteran insectlarvae black cutworm (Agrotis ipsilon), fall armyworm (Spodopterafrugiperda), beet armyworm (Spodoptera exigua), tobacco budworm(Heliothis virescens), and corn earworm (Helicoverpa zea). However,Vip does not show any homology with the known crystallineinsecticidal proteins. This structural dissimilarity is indicative of apossible divergent insecticidal mechanism than the other known Bt- toxins. These observed structural divergences of Vip with Bt toxinsmake them an ideal candidate for deployment in insect managementprograms together with the other category of Bt-toxins.

Proteinase inhibitors

The protease inhibitor (PIs) proteins are natural antagonist’sproteins and they are quite common in all life forms [24]. In plantsthey play defensive mechanisms against phytophagous insects andmicroorganisms. Most PIs interact with their target proteases bycontact with the active (catalytic) site of the protease, resulting in theformation of a stable protease-inhibitor complex that is incapableof enzymatic activity [25]. Proteinases inhibitors are classifiedaccording to their catalytic mechanisms into four classes (1) serineproteinases, with a serine and histidine; (2) cysteine proteinases,with a cysteine; (3) aspartic proteinases, with an aspartate groupand (4) metalloproteinases, with a metallic ion (Zn⁺², Ca⁺² or Mn⁺²)[26]. In plants, proteinase inhibitors have different role, such asstorage proteins, as regulators of endogenous proteolytic activity[27], as participants in many developmental processes, includingprogrammed cell death and as components associated with theresistance of plants against insects and pathogens [28]. They may besynthesized constitutively during normal development or may beinduced in response to insect and pathogen attacks [27].

Transgenic plants expressing PI genes began when Hilder etal. [29] transformed tobacco plants with the trypsin inhibitor gene(CpTI) of Cowpea which showed reduced growth and mortality inlarvae of Heliothis virescens (bollworm). In the 1990s Gatehouse et al.[30] transformed tobacco plants with the trypsin inhibitor gene of soya(Kunitz family) (SBTI), which showed a high inhibitory effect on thelarvae of H. virescens. Many PIs were expressed in transgenic plantswhich conferred the protection against Chrysodeixis eriosoma (greenmeasuring worm), Sesamia inferens (pink stem borer), Spodopteralitura (tobacco budworm), Nilaparvata lugens (brown planthopper),Sitotroga cerealella (Angoumoid grain moth), Tribolium castaneum(brown flower beetle) [30-32]. Considering all the evidence which hasappeared in the text, the effect that the PIs have on insects is evident;however, negative effects have also been shown on beneficial insects[33]. Later it was realized that the insects overcome enzyme inhibitoryproperty (PIs) by altering their enzyme specificity by mutations, thusdampening the hopes of using enzyme inhibitors for transgenic crops[10].

α-Amylase inhibitors

α-Amylases (α-1, 4-glucan-4-glucanohydrolases) are widespreadhydrolytic enzymes found in microorganisms, animals and plants.They catalyze the initial hydrolyses of α-1,4-linked sugar polymers,such as starch and glycogen into shorter oligosaccharides, animportant step towards transforming sugar polymers into single unitsthat can be assimilated by the organism. Higher plants and animalsproduce a large number of different protein inhibitors of α-amylasesin order to regulate the activity of these enzymes [34]. However theseα-AIs are used to generate transgenic plants that are resistant againstinsect pest [35]. The expression of the α-Al gene encoding protein inplant system, such as pea (Pisum sativum L.) and azuki bean (Vignaanguralis L.) showed promising effect against bruchid beetle pests (Coleoptera: Bruchidae) [36]. Rye α-amylase inhibitor expressed intransgenic tobacco seeds (Nicotiana tabacum) caused 74% mortalityin Anthonomus grandis first instar larvae when transgenic seed flourmixture used in artificial diet [37]. However these inhibitors used asplant resistance factors are effective against Coleoptera order andthey cannot be used to control different orders of agriculture insectpests [38-40].

Arcelins

Post harvest loss due to insect pests largely affects the overallfood grain production and consumption and it is estimated to be13%. Arcelins are antinutritional insecticidal seed storage proteins,found in the wild bean Phaseolus vulgaris, which have been shownto prevent infestation by post harvest insect pests such as bruchidbeetles [41]. Amino acid sequence comparison shows that arcelinsbelong to the bean lectin-like family which includes the two typesof phytohemagglutin subunits (PHA-L and PHA-E) and α-amylaseinhibitors [42]. Although the members of this protein family displaysimilar tertiary structures, they differ in their biochemical properties,glycosylation patterns, quaternary structure and sugar bindingspecificities [43]. Insecticidal properties of arcelins variants towardbruchid pests Z. subfasciatus has been reported [44], which is knownto be one of the most important pests of stored beans.

Ribosome-inactivating proteins

Ribosome-inactivating proteins (RIPs) are a group of plantproteins that are capable of specifically and irreversibly inactivatingeukaryotic ribosome’s and inhibits protein translation, which playsan important role in plant defense and hence can be exploited inplant protection [45]. Over a hundred RIPs have been isolated fromvarious plants and bacteria with varying degrees of toxicity. RIPs aresubdivided on the basis of their molecular structure into three distinctgroups. Type I RIPs are monomeric proteins of approximately 30kDa which possess RNA N-glycosidase enzymatic activity. Type IIis a heterodimer consisting of an A-chain with RNA N-glycosidaseactivity associated to one or several B-chain(s) of approximately 35kD, functionally equivalent to the Type I polypeptide [46] linked toa B subunit. The B-subunit is a lectin-like peptide that has strongaffinity for sugar moieties displayed on the surface of cells and helpsto promote translocation through the plasma membrane [47]. WhileType III is synthesized as inactive precursors (Pro RIPs) that requireproteolytic processing events to form an active RIP [48]. Ricin, Abrinand Modeccin are well known examples of RIPs, which irreversiblyinactivate ribosomes by removing a specific adenine from a highlyconserved tetra-nucleotide loop present in the large ribosomalsubunit [49]. Some of these RIPs, such as ricin (type-II RIPs) havehigh toxicity affect against a variety of insects, although these effectsare variable on different insect orders [50]. More recently, Shahidi-Noghabi et al. [51] demonstrated that expression of Sambucus nigeragglutinin (SNA-I, type-II RIPs) from elderberry bark in transgenictobacco has a deleterious effect on two important insect pests, thetobacco aphid Myzus nicotianae and the beet armyworm Spodopteraexigua. Although the biochemical properties of the RIPs are wellstudied, but their exact mechanisms of action at the tissue level ofRIPs-ingested insects are not well understood [51-53]. In view of theseargued apprehensions on the sustainability of Bt crops, and other narrow range of biological pest control molecules, alternative sourcesof potential insecticidal gene products need to be explored whichshows wide range of insect control. Lectins from different sources andclasses are an attractive alternative candidate in transgenic-based pestcontrol strategies [45].

Lectins

Lectins are a class of proteins of non-immune origin that possessat least one non-catalytic domain that specifically and reversibly bindto mono- or oligosaccharide [54]. A typical lectin is multivalent;and because of its specific carbohydrate binding property it is ableto agglutinate cells. Lectins are extensively distributed in natureand several hundred of these molecules have been isolated so farfrom plants, microorganisms, fungi and vertebrates, includingmammals [55]. Because of their unique ability to bind to certainspecific membrane glycoproteins, some of lectin exerts proliferative,antiproliferative, immunomodulatory effects. Such propertiesexhibited by lectins made them useful tools in diverse areas like cancerdiagnosis and therapeutics, virology, structural biology, separationtechnology, bacterial typing and insect toxicology [56,57].

Lectins with insecticidal property

Several biological functions of lectins have been reported [58]among them anti-insect activities have received particular attentionin the pest management strategies [59,60]. Lectins basically bind toglycosylated proteins, as the glycosylation of protein is a key posttranslational event. In organisms there are two types of glycosylationpattern occur such as, N-linked and O-linked based on the linkage ofcarbohydrate moiety to the protein backbone. N-glycans are linked toAsn residues of protein backbone via N-acetyl glucosamine, whereasO-glycans linked to hydroxyl group of serine or threonine residuesvia N-acetyl galactosamine. N- glycans and O- glycans are profuselyfound in insects [61,62], because of lectins unique ability to bind tocertain specific membrane glycoproteins in the insects made themvaluable tool in the pest management science. There are substantialevidences that, lectins bind to gut surface glycans of gut epithelial cellsor bind to secretory gut proteins causing physiological imbalance,resulting in immunomodulatory effect such as apoptosis [60,63,64].However, the molecular mechanism of lectin induced toxicity ininsects has not been understood in detail [60,64].

Many lectins are highly toxic for phytophagous insects; the useof lectins in transgenic plants has yielded positive results on insect’spest belonging to different orders such as Lepidoptera, Coleoptera,Diptera and Hemiptera. Among lectins, Plant originated N-glycanspecific lectins received greater attention due to their toxic effectsagainst broad range of economically important insect pests [62].The first N-glycan specific lectin from plant origin, Galanthus nivalisagglutinin (GNA) has showed toxic effects against hemipterans andother economically important insect pests. GNA has been successfullyengineered into a variety of crops including sugarcane, rice, wheat,potatoes or tobacco to give them a higher resistance against differentorder insect pest. Followed by GNA many plant lectins such as, Wheatgerm agglutinin (WGA), Pisum Sativum Agglutinin (PSA), Phaseolusvulgaris Agglutinin (PHA) and Allium sativum lectin (ASAL) weresuccessfully expressed in important agriculture crops and they have been shown to exert deleterious effects on a range of important pestinsects [60,64]. Unlike the N- linked mannose binding lectins, verylittle is known about O-glycan specific lectin. Recently O-glycan(Gal/GalNAc) specific lectins are reported for their toxic effects oninsect pests. Among plant lectin, Amaranthus caudatus is the onlyreported O-linked glycan specific lectin employed in transgenicplants shown to be effective against insects [65,66]. Compared toplant and animal lectins, very little information is available on lectinsfrom fungal origin [67,68]. In recent years, mushroom and otherfungal lectins of different carbohydrate specificity have got muchattention in agriculture field as a bio-insecticide. All these studiesshows that lectins from different origins, potential to be exploited incrop protection against various insect pests. Thus the use of lectinsfrom different origin have proved to be more efficient ways to controlchewing and sap-sucking insect pests on agriculturally importantcrops.