decades, important progress has been made in the study of the activity of plant lectins against pathogens, nematodes and especially insect pests (Ma et al., 2010; Peumans and Van Damme, 1995; Vasconcelos and Oliveira, 2004). The best-characterized family of plants lectins are Fabaceae, Poaceae and Solanaceae; especially some of leguminous seeds have a remarkable amount of lectin. Different food crops such as tomato, wheat, rice, potato, soybean and bean contain lectins. The great majority of the plant lectins are present in seed cotyledons but a lot of them are also found in the protein bodies such as roots, leaf, stems, rhizomes, bark, bulbs, tubers, corms, fruits, flowers, ovaries, phloem sap, latex, nodule and

beetle)

*nubilaris*

(colorado potato beetle)

*nubilaris*

aphid)

*nubilaris*

*nubilaris*

*nubilaris*

*nubilaris*

plant bug)

a Sugar specificity is represented by the best monosaccharide inhibitor.

Table 1. Plant lectins with oral toxicity to insects (Adapted from Vasconcelos et al., 2004)

decades, important progress has been made in the study of the activity of plant lectins against pathogens, nematodes and especially insect pests (Ma et al., 2010; Peumans and Van Damme, 1995; Vasconcelos and Oliveira, 2004). The best-characterized family of plants lectins are Fabaceae, Poaceae and Solanaceae; especially some of leguminous seeds have a remarkable amount of lectin. Different food crops such as tomato, wheat, rice, potato, soybean and bean contain lectins. The great majority of the plant lectins are present in seed cotyledons but a lot of them are also found in the protein bodies such as roots, leaf, stems, rhizomes, bark, bulbs, tubers, corms, fruits, flowers, ovaries, phloem sap, latex, nodule and

Lectins and Their Roles in Pest Control 213

Obtained results from the effects of *Canavalia ensiformis* agglutinin (Con A) and *Galantus nivalis* agglutinin (GNA) on the developmental period and fecundity of the peach-potato aphid, *Myzus persicae* showed that adult survival was not significantly altered, but both lectins adversely affected total fecundity and developmental period (Sauvion et al., 1996). Later, the same assay was performed to evaluate the efficiency of Con A in pea aphid, *Acyrthosiphon pisum*. Results showed that Con A has highly significant toxic effects on *A. pisum*. It also induced remarkable effects on the structure of midgut epithelial cells of this aphid (Sauvion et al., 2004). These results clearly show that plants lectins play a crucial role

Among plant lectins presented in table (1) as entomotoxic lectins some of which especially GNA, WGA, PSA, PHA and ConA were more successfully expressed in a range of crops such as Tomato, Rice, Sugarcane, Tobacco, Maize, Mustard and Arabidopsis (Table 1) and they have been shown to exert deleterious effects on a range of important pest insects (Maddock et al., 1991; Kanrar et al., 2002; Boulter et al., 1990c; Bell et al., 1999, 2001; Down et al., 2001 ; Maqbool et al., 2001; Sun et al., 2002; Wu et al., 2002 ; Setamou et al., 2002; Down et al., 1996; Fitches et al., 1997, 2001; Rao et al., 1998; Foissac et al., 2000). Currently, the two major groups of plant derived genes used to confer insect resistance on crops are lectins and inhibitors of digestive enzymes (proteases and amylase inhibitors). Lectins have been introduced into crops genomes and are now being tested in field conditions (Gatehouse et al., 1993; Hilder et al., 1987; Hilder et al., 1999; Carlini et al., 2002; Schuler et al., 1998; Ranjeker et al., 2003; Schnepf & whitely, 1981; Smith & Boyko, 2006; Christou et al., 2006; Wang, 2006; Zhao, 2006; Ferry, 2006). Also, for the first time Jjanhong et al (2003) reported that transgenic tobacco expressing *Pinellia ternata* agglutinin (*pta*) gene induced enhance level of resistance to *M. persicae*. Additionally, crops have been engineered to express a range of insect-plant resistance (Table 2), and have been shown to confer enhanced levels of resistance to different order of insect pests including lepidopteran (Gatehouse et al., 1997), and homopteran (Down et al., 1996; Gatehouse et al., 1996), when expressed in wheat. Transgenic plants technology or genetically modified (GM) crops can be a useful tool to produce resistant crops; by introducing novel resistance genes into plants thus it provides a sustainable alternative to the control of pest

insects and pathogens by pesticides (Gatehouse et al., 1997; 1999; Gray et al*.*, 2003).

Down et al., 1996; Bandyopadhyay et al., 2001).

**6. Fungal lectins with insecticidal activity**

On the whole, transgenic plants expressing high levels of lectins exhibited some degree of resistance to the target insects. Some of lectins such as GNA, WGA and ConA have been succefuly expressed in plants to confer resistence pest insects (Table 2) (Powell et al., 1995;

Mushrooms contain various potential interesting proteins, including lectins in their organs such as mycelium, spores and fruiting bodies (Wang et al., 1998; 2002; Ng, 2004; Nelson & Cox, 2005). For many years, all investigations were only focused on plant lectins with insecticidal activity. Even though lectins are found in many kinds of organisms such as fungi, but there is little information about their toxicity on phytophagus insects. Therefore, at present our knowledge about insecticidal activity of fungal lectins is limited. Due to lack of sufficient knowledge, one of the aims of this chapter is to introduce and highlight the

in plant resistance against insect pests.

**5. Transgenic plants with insecticidal lectin gene**

even in nectar (Van Damme et al., 1998). Plant lectins function as storage proteins and they have been implicated in defence mechanisms against phytophagus insects (Powell et al., 1993; Peumans & Van Damme, 1995; Van Damme et al., 1998; Rudiger & Gabius, 2001; Gatehouse et al., 1995; Powell, 2001; Carlini & Grossi-de-Sa´, 2002; karimi et al., 2010). Various plants lectins have already been found to be toxic towards important members of insect orders, including Coleoptera (Gatehouse et al., 1984), Lepidoptera (Czapla & Lang, 1990) and Homoptera (Powell et al., 1993; Sauvion et al., 1996) (Table 1). The first lectin to be purified on a large scale and was available on a commercial basis was Concanavalin A; which is now the most well- known lectin to control of some pest insects (Fig. 1A). Now a wide range of plant lectins have been successfully examined for their negative effects on the life parameters of some economically pest insects (Gatehouse et al., 1995; Powell, 2001; Foissac et al., 2000; Couty et al., 2001b; Sauvion *et al*., 2004; karimi et al., 2007; Shahidi-Noghabi et al., 2008, 2009) (Table 1).

Fig. 1. **(A).** *Canavalia ensiformis*, or Jack-bean (Common name), is a legume plant in the Fabaceae family of which is used for animal fodder and human nutrition, especially in Brazil. It is also the source of concanavalin A lectin. **(B)** *Galanthus nivalis* or snowdrop (Common name), is the best-known and most widespread representative plant in the Amaryllidaceae family. (Figures from Wikipedia, (A) *Canavalia ensiformis*, (B) *Galanthus nivalis*)

Three mannose-binding specific lectins include *Galanthus nivalis* (GNA), *Narcissus pseudonarcissus* (NPA) and *Allium sativum*(ASA) were assayed in artificial diets for their toxic and growth-inhibitory effects on nymphal development of the peach-potato aphid, *Myzus persicae*. Results showed that the snowdrop lectin (GNA) was the most toxic, with an induced nymphal mortality of 42% at 1500 μg/ml and an median insect toxicity value IC50 (50% growth inhibition) of 630 μg/ml (Fig. 1B). But daffodil lectin (NPA) and a garlic lectin (ASA) induced no significant mortality in the range of 10–1500 μg/ml (Sauvion et al., 1996).

even in nectar (Van Damme et al., 1998). Plant lectins function as storage proteins and they have been implicated in defence mechanisms against phytophagus insects (Powell et al., 1993; Peumans & Van Damme, 1995; Van Damme et al., 1998; Rudiger & Gabius, 2001; Gatehouse et al., 1995; Powell, 2001; Carlini & Grossi-de-Sa´, 2002; karimi et al., 2010). Various plants lectins have already been found to be toxic towards important members of insect orders, including Coleoptera (Gatehouse et al., 1984), Lepidoptera (Czapla & Lang, 1990) and Homoptera (Powell et al., 1993; Sauvion et al., 1996) (Table 1). The first lectin to be purified on a large scale and was available on a commercial basis was Concanavalin A; which is now the most well- known lectin to control of some pest insects (Fig. 1A). Now a wide range of plant lectins have been successfully examined for their negative effects on the life parameters of some economically pest insects (Gatehouse et al., 1995; Powell, 2001; Foissac et al., 2000; Couty et al., 2001b; Sauvion *et al*., 2004; karimi et al., 2007; Shahidi-

Three mannose-binding specific lectins include *Galanthus nivalis* (GNA), *Narcissus pseudonarcissus* (NPA) and *Allium sativum*(ASA) were assayed in artificial diets for their toxic and growth-inhibitory effects on nymphal development of the peach-potato aphid, *Myzus persicae*. Results showed that the snowdrop lectin (GNA) was the most toxic, with an induced nymphal mortality of 42% at 1500 μg/ml and an median insect toxicity value IC50 (50% growth inhibition) of 630 μg/ml (Fig. 1B). But daffodil lectin (NPA) and a garlic lectin (ASA) induced no significant mortality in the range of 10–1500 μg/ml (Sauvion et al., 1996).

Fig. 1. **(A).** *Canavalia ensiformis*, or Jack-bean (Common name), is a legume plant in the Fabaceae family of which is used for animal fodder and human nutrition, especially in Brazil. It is also the source of concanavalin A lectin. **(B)** *Galanthus nivalis* or snowdrop (Common name), is the best-known and most widespread representative plant in the Amaryllidaceae

family. (Figures from Wikipedia, (A) *Canavalia ensiformis*, (B) *Galanthus nivalis*)

Noghabi et al., 2008, 2009) (Table 1).

**A B**

Obtained results from the effects of *Canavalia ensiformis* agglutinin (Con A) and *Galantus nivalis* agglutinin (GNA) on the developmental period and fecundity of the peach-potato aphid, *Myzus persicae* showed that adult survival was not significantly altered, but both lectins adversely affected total fecundity and developmental period (Sauvion et al., 1996). Later, the same assay was performed to evaluate the efficiency of Con A in pea aphid, *Acyrthosiphon pisum*. Results showed that Con A has highly significant toxic effects on *A. pisum*. It also induced remarkable effects on the structure of midgut epithelial cells of this aphid (Sauvion et al., 2004). These results clearly show that plants lectins play a crucial role in plant resistance against insect pests.

#### **5. Transgenic plants with insecticidal lectin gene**

Among plant lectins presented in table (1) as entomotoxic lectins some of which especially GNA, WGA, PSA, PHA and ConA were more successfully expressed in a range of crops such as Tomato, Rice, Sugarcane, Tobacco, Maize, Mustard and Arabidopsis (Table 1) and they have been shown to exert deleterious effects on a range of important pest insects (Maddock et al., 1991; Kanrar et al., 2002; Boulter et al., 1990c; Bell et al., 1999, 2001; Down et al., 2001 ; Maqbool et al., 2001; Sun et al., 2002; Wu et al., 2002 ; Setamou et al., 2002; Down et al., 1996; Fitches et al., 1997, 2001; Rao et al., 1998; Foissac et al., 2000). Currently, the two major groups of plant derived genes used to confer insect resistance on crops are lectins and inhibitors of digestive enzymes (proteases and amylase inhibitors). Lectins have been introduced into crops genomes and are now being tested in field conditions (Gatehouse et al., 1993; Hilder et al., 1987; Hilder et al., 1999; Carlini et al., 2002; Schuler et al., 1998; Ranjeker et al., 2003; Schnepf & whitely, 1981; Smith & Boyko, 2006; Christou et al., 2006; Wang, 2006; Zhao, 2006; Ferry, 2006). Also, for the first time Jjanhong et al (2003) reported that transgenic tobacco expressing *Pinellia ternata* agglutinin (*pta*) gene induced enhance level of resistance to *M. persicae*. Additionally, crops have been engineered to express a range of insect-plant resistance (Table 2), and have been shown to confer enhanced levels of resistance to different order of insect pests including lepidopteran (Gatehouse et al., 1997), and homopteran (Down et al., 1996; Gatehouse et al., 1996), when expressed in wheat. Transgenic plants technology or genetically modified (GM) crops can be a useful tool to produce resistant crops; by introducing novel resistance genes into plants thus it provides a sustainable alternative to the control of pest insects and pathogens by pesticides (Gatehouse et al., 1997; 1999; Gray et al*.*, 2003).

On the whole, transgenic plants expressing high levels of lectins exhibited some degree of resistance to the target insects. Some of lectins such as GNA, WGA and ConA have been succefuly expressed in plants to confer resistence pest insects (Table 2) (Powell et al., 1995; Down et al., 1996; Bandyopadhyay et al., 2001).

#### **6. Fungal lectins with insecticidal activity**

Mushrooms contain various potential interesting proteins, including lectins in their organs such as mycelium, spores and fruiting bodies (Wang et al., 1998; 2002; Ng, 2004; Nelson & Cox, 2005). For many years, all investigations were only focused on plant lectins with insecticidal activity. Even though lectins are found in many kinds of organisms such as fungi, but there is little information about their toxicity on phytophagus insects. Therefore, at present our knowledge about insecticidal activity of fungal lectins is limited. Due to lack of sufficient knowledge, one of the aims of this chapter is to introduce and highlight the

Lectins and Their Roles in Pest Control 215

*melanogaster*) and homopteran (*Myzus persicae* and *Acyrthosipon pisum* (Trigueros et al., 2003; Karimi et al., 2008). Later, effect of this edible wild mushroom (Fig. 1) was evaluated on *M. persicae* aphid by Karimi et al (2008) and obtained results showed that the sub lethal dose of XCL (<50 μg/ml) has significant effects on biological parameters (larval weight, developmental period and fecundity) of *M. persicae* in compare with sub lethal dose of Con A (<50 μg/ml) on biological parameters of this aphid under laboratory conditions (Abbott, 1925;

Recently, the results from insecticidal properties of *Sclerotinia sclerotiorum* agglutinin (SSA) and its interaction with pea aphid, *Acyrthosiphon pisum* tissues and cells showed that this fungal lectin has high mortality on *A. pisum* with a median insect toxicity value (IC50) of 66 μg/ml. Also these results revealed that SSA has significant cell toxicity on *A. pisum* midgut tract and its brush border cells (Hamshou et al., 2010) (Fig. 2). Moreover, a purified lectin from *Rhizoctonia solani* agglutinin (RSA), which exhibits specificity towards N-acetyl/galactosamine, was shown to exert deleterious effects on the growth, developmental time, survival and the

More recently, another new mannose- specific lectin with insecticidal activity has been successfully purified from *Penicillum chrysogenum* (PeCL). This lectin has high insecticidal activity on aphids, especially to *M. persicae* in comparison with well-known plant lectins,

Consequently until now, several mushroom lectins including, *Xerocomus chrysenteron* lectin (XCL), *Penicillum chrysogenum* lectin ((PeCL) and *Sclerotinia sclerotium* agglutinin (SSA) have shown greater potential effects on some important pest insects such as *Myzus persicae, Acyrthosipon pisum* and *Spodoptera littoralis* compare to well known lectins such as ConA and GNA (Trigueros et al., 2003; karimi et al., 2006, 2007, 2008; Hamshou et al., 2010; Francis et al., 2011). As a result, it is concluded that fungal lectin will be able to confer enhanced level

Investigation on the lectin toxicity at the cellular level in insects were initiated 24 years ago, when Gatehouse et al. (1984) firstly reported the binding of *Phaseolus vulgaris* lectin (PHA) to midgut epithelial cells of the cowpea weevil, *Callosobruchus maculatus*. In fact, the mode of action for each lectin at the tissue level of ingested lectin organisms is depended on presence of appropriate carbohydrate moieties on the organ surface and the ability of lectin to bind

In general, the action mechanism of the lectin at cellular level of ingested lectin by insects showed that binding of the lectin to the midgut tract causing disruption of the epithelial cells including elongation of the striated border microvilli, swelling of the epithelial cells into the lumen of the gut lead to complete closure of the lumen, permeability of cell membrane to allow the harmful substances penetrations from lumen towards haemolymph and impaired nutrient assimilation by cells, allowing absorption of potentially harmful substances from lumen into circulatory system, fat bodies, ovarioles and throughout the haemolymph (Gatehouse et al., 1984; Powell et al., 1998; Habibi et al., 1998; 2000; Fitches et al., 1998; 2001b; Sauvion et al., 2004; Majumder et al., 2005). This information gave further support to previous suggestions that the XCL lectins disrupt midgut cells (Francis et al., 2003; Karimi et al., 2008, 2009). Lectins are highly specific for binding to oligosaccharides, hence if specific carbohydrate is in the surface of tissue it can bind to them and it is believed

larval weight of the cotton leaf worm, *Spodoptera littoralis* (Hamshou et al., 2010).

ConA. (Francis et al, 2011; karimi et al., 2006, 2007 and 2008), (Table 3 and Fig. 3B).

of resistance in plants against their phytophagous insects.

them (Fitches et al., 2001a; 2001b).

**7. Action mechanism of lectin at the tissue level of insects**

Karimi et al., 2008), (Table 3 and Fig. 3A).

fungal lectins with insecticidal activity. Recently, important progress is made in the study of the fungal lectins against pathogens, especially pest insects (karimi et al., 2007 and 2008; Hamshou et al., 2010; Francis et al., 2011). Many lectins have been derived from different fungi and partially isolated and characterized for their effects on mammalian physiology as antitumor and anticancer, but there is little information on their role on phytophagous insects (Wang et al., 2002; Trigueros et al., 2003, Karimi et al., 2008).

a: For lectin abbreviations see Table 1.

c: First demonstration of insect enhanced resistance of transgenic plants expressing a foreign lectin.

Table 2. Transgenic plants with lectin genes to confer resistance against insects (Adapted from Vasconcelos et al., 2004)

Some lectins from fungi including *Xerocomus chrysenteron* (XCL), *Arthrobotrys oligospora* (AOL) and *Agaricus bisprous* (ABL) have been isolated and all are well known for their reversible antiproliferative effects. But, only XCL has shown significant effects and exhibited a higher insecticidal activity on the some orders of insect pests, such as dipteran (*Drosophila*

fungal lectins with insecticidal activity. Recently, important progress is made in the study of the fungal lectins against pathogens, especially pest insects (karimi et al., 2007 and 2008; Hamshou et al., 2010; Francis et al., 2011).  Table 2. Transgenic plants with lectin genes to confer resistance against insects (Adapted

Some lectins from fungi including *Xerocomus chrysenteron* (XCL), *Arthrobotrys oligospora* (AOL) and *Agaricus bisprous* (ABL) have been isolated and all are well known for their reversible antiproliferative effects. But, only XCL has shown significant effects and exhibited a higher insecticidal activity on the some orders of insect pests, such as dipteran (*Drosophila*

*Diatraea saccharalis (*sugarcane borer)

insects (Wang et al., 2002; Trigueros et al., 2003, Karimi et al., 2008).

Maize WGA *Ostrinia nubilaris;Diabrotica*

Potato GNA *Aphidius ervi (*parasitoid of *M.*

Rice GNA *Nephotettix virescens* (green

Rice GNA *Cnaphalocrocis medinalis* (rice

Rice GNA *Laodelphax striatellus* (rice small

a: For lectin abbreviations see Table 1.

from Vasconcelos et al., 2004)

Sugarcane GNA *Eoreuma loftini (*Mexican rice borer*);*

Sugarcane GNA *Parallorhogas pyralophagus (*parasitoid

*persicae)*

leafhopper)

of E. loftini)

*melanogaster*) and homopteran (*Myzus persicae* and *Acyrthosipon pisum* (Trigueros et al., 2003; Karimi et al., 2008). Later, effect of this edible wild mushroom (Fig. 1) was evaluated on *M. persicae* aphid by Karimi et al (2008) and obtained results showed that the sub lethal dose of XCL (<50 μg/ml) has significant effects on biological parameters (larval weight, developmental period and fecundity) of *M. persicae* in compare with sub lethal dose of Con A (<50 μg/ml) on biological parameters of this aphid under laboratory conditions (Abbott, 1925; Karimi et al., 2008), (Table 3 and Fig. 3A).

Recently, the results from insecticidal properties of *Sclerotinia sclerotiorum* agglutinin (SSA) and its interaction with pea aphid, *Acyrthosiphon pisum* tissues and cells showed that this fungal lectin has high mortality on *A. pisum* with a median insect toxicity value (IC50) of 66 μg/ml. Also these results revealed that SSA has significant cell toxicity on *A. pisum* midgut tract and its brush border cells (Hamshou et al., 2010) (Fig. 2). Moreover, a purified lectin from *Rhizoctonia solani* agglutinin (RSA), which exhibits specificity towards N-acetyl/galactosamine, was shown to exert deleterious effects on the growth, developmental time, survival and the larval weight of the cotton leaf worm, *Spodoptera littoralis* (Hamshou et al., 2010).

More recently, another new mannose- specific lectin with insecticidal activity has been successfully purified from *Penicillum chrysogenum* (PeCL). This lectin has high insecticidal activity on aphids, especially to *M. persicae* in comparison with well-known plant lectins, ConA. (Francis et al, 2011; karimi et al., 2006, 2007 and 2008), (Table 3 and Fig. 3B).

Consequently until now, several mushroom lectins including, *Xerocomus chrysenteron* lectin (XCL), *Penicillum chrysogenum* lectin ((PeCL) and *Sclerotinia sclerotium* agglutinin (SSA) have shown greater potential effects on some important pest insects such as *Myzus persicae, Acyrthosipon pisum* and *Spodoptera littoralis* compare to well known lectins such as ConA and GNA (Trigueros et al., 2003; karimi et al., 2006, 2007, 2008; Hamshou et al., 2010; Francis et al., 2011). As a result, it is concluded that fungal lectin will be able to confer enhanced level of resistance in plants against their phytophagous insects.

#### **7. Action mechanism of lectin at the tissue level of insects**

Investigation on the lectin toxicity at the cellular level in insects were initiated 24 years ago, when Gatehouse et al. (1984) firstly reported the binding of *Phaseolus vulgaris* lectin (PHA) to midgut epithelial cells of the cowpea weevil, *Callosobruchus maculatus*. In fact, the mode of action for each lectin at the tissue level of ingested lectin organisms is depended on presence of appropriate carbohydrate moieties on the organ surface and the ability of lectin to bind them (Fitches et al., 2001a; 2001b).

In general, the action mechanism of the lectin at cellular level of ingested lectin by insects showed that binding of the lectin to the midgut tract causing disruption of the epithelial cells including elongation of the striated border microvilli, swelling of the epithelial cells into the lumen of the gut lead to complete closure of the lumen, permeability of cell membrane to allow the harmful substances penetrations from lumen towards haemolymph and impaired nutrient assimilation by cells, allowing absorption of potentially harmful substances from lumen into circulatory system, fat bodies, ovarioles and throughout the haemolymph (Gatehouse et al., 1984; Powell et al., 1998; Habibi et al., 1998; 2000; Fitches et al., 1998; 2001b; Sauvion et al., 2004; Majumder et al., 2005). This information gave further support to previous suggestions that the XCL lectins disrupt midgut cells (Francis et al., 2003; Karimi et al., 2008, 2009). Lectins are highly specific for binding to oligosaccharides, hence if specific carbohydrate is in the surface of tissue it can bind to them and it is believed

Lectins and Their Roles in Pest Control 217

 Fig. 3. (**A**) *Xercomus chrysenteron* fungus naturally growth in forest. It is a small, edible wild mushroom in the Boletaceae family and has a cosmopolitan distribution, concentrated in cool-temperate to subtropical regions. (**B**) *Penicillums chrysogenum* fungus growth in medium culture. It can be found on salted food products as well as indoor environments, especially in damp or water damaged buildings. This fungus is in the Tricocomaceae family. To date is used as anti bacterial disease. (Figures from Wikipedia, (A) *Xercomus chrysenteron,*

that these cell-surface receptors are responsible for lectin binding. Thus, it could be concluded that the action mechanism of various lectins at the cellular levels of insects differs between different insect species (Habibi et al., 2000; Fitches et al., 2001a; Sauvion et al., 2004; Karimi et al., 2009). Consequently, the action mechanism of the lectin at the cellular level of

In some case lectin have an indirect remarkable effects; such as interaction with virus

In general some of insects such as aphids transmit virus from infected plant to non infected plants. Some lectins such as mannose-binding lectins are able to bind to carbohydrate on micro-organisms. Circulatory viruses contain numerous *N*-linked glycosylation sites on their surface cells. Many of these sites contain high-mannose glycans which could interact with mannose-binding lectin such as ConA (Gray et al., 1999; Brisson and Stern, 2006; Hogenhout et al., 2008; Desoignies, 2008; Thielens et al., 2002; Pereira et al., 2008; Naidu et

insect are not clearly elucidated yet and the information is scarce.

**8. Indirect effects of lectins on pests control**

**A B**

**8.1 Interaction with virus transmission**

transmission and synergistic effects on the other proteins.

al., 2004; Dimitrov, 2004; Garret et al., 1993; Wei, 2007).

(B) *Penicillums chrysogenum*)

Fig. 2. Effect of different concentrations of SSA on insect midgut CF-203 cells. Cells were incubated for 4 days at 27◦C. (A) Control, (B) Treated cells with 25 mg/ml SSA, (C) SSA toxicity towards CF-203 midgut cells. Cell toxicity was measured using an MTT assay after 4 days of exposure to SSA at various concentrations. Data are presented as mean percentages of cell toxicity ± SEM compared to the control, and based on four repeats and the experiments were repeated two or three times. Values with a different letter are significantly different after a post hoc Tukey Kramer test (p ¼ 0.05) (Figure from Hamshou et al., 2010).

Table 3. Fungal lectins with insecticidal activity (Karimi et al., 2011)

Fig. 2. Effect of different concentrations of SSA on insect midgut CF-203 cells. Cells were incubated for 4 days at 27◦C. (A) Control, (B) Treated cells with 25 mg/ml SSA, (C) SSA toxicity towards CF-203 midgut cells. Cell toxicity was measured using an MTT assay after 4 days of exposure to SSA at various concentrations. Data are presented as mean percentages

experiments were repeated two or three times. Values with a different letter are significantly different after a post hoc Tukey Kramer test (p ¼ 0.05) (Figure from Hamshou et al., 2010).

Peach, potato,

Trigueros, et al.,

Karimi et al., 2008

Hamshou et al.,

Hamshou et al.,

Francis et al.,

2003 ;

2010b

2010a

2011

Pea

Pea

cotton

Pea

Peach, potato,

of cell toxicity ± SEM compared to the control, and based on four repeats and the

**Lectin (fungal source) Insect Host Reference**

 *M. persicae, Acyrthosipon*

*Drosophila melanogaster*

*Acyrthosipon pisum*

*Spodoptera littoralis*

*M. persicae, Acyrthosipon*

*pisum*

*pisum*

Table 3. Fungal lectins with insecticidal activity (Karimi et al., 2011)

**N-acetyl-D-galactosamine**

**XCL(***Xerocomus chrysenteron***)**

**SSA** (*Sclerotinia sclerotium*)

**RSA** (*Rhizoctonia solani*

**Mannose specific PeCL(***penicillum chrysogenum* **)**

agglutinin)

**specific**

that these cell-surface receptors are responsible for lectin binding. Thus, it could be concluded that the action mechanism of various lectins at the cellular levels of insects differs between different insect species (Habibi et al., 2000; Fitches et al., 2001a; Sauvion et al., 2004; Karimi et al., 2009). Consequently, the action mechanism of the lectin at the cellular level of insect are not clearly elucidated yet and the information is scarce.

#### **8. Indirect effects of lectins on pests control**

In some case lectin have an indirect remarkable effects; such as interaction with virus transmission and synergistic effects on the other proteins.

#### **8.1 Interaction with virus transmission**

In general some of insects such as aphids transmit virus from infected plant to non infected plants. Some lectins such as mannose-binding lectins are able to bind to carbohydrate on micro-organisms. Circulatory viruses contain numerous *N*-linked glycosylation sites on their surface cells. Many of these sites contain high-mannose glycans which could interact with mannose-binding lectin such as ConA (Gray et al., 1999; Brisson and Stern, 2006; Hogenhout et al., 2008; Desoignies, 2008; Thielens et al., 2002; Pereira et al., 2008; Naidu et al., 2004; Dimitrov, 2004; Garret et al., 1993; Wei, 2007).

Lectins and Their Roles in Pest Control 219

Bandyopadhyay S., Roy A. & Das S., 2001. Binding of garlic (*Allium sativum*) leaf lectin to the

Baldwin I. T., Sims C. L. & Kean S. E., 1990. The reproductive consequences associated with

Bell H. A., Fitches E. C., Down R. E., Marris G. C., Edwards J. P., Gatehouse J. A. &

Bell H. A., Fitches E. C., Down R. E., Ford L., Marris G.C., Edwards J. P., Gatehouse J.A.,

*pennicornis* (Hymenoptera: Eulophidae). *Pest Manage. Sci*., 57(1), 57-65. Bell H. A., Kirkbride-Smith A. E., Marris G. C., Edwards J. P. & Gatehouse A. M. R., 2004.

Boulter D., Edwards G. A., Gatehouse A. M. R., Gatehouse J. A. & Hilder V. A., 1990.

insect resistance genes in transgenic tobacco plants. *Crop Prot*., 9(5), 351-354. Brisson, J. A., Stern, D. L., 2006. The pea aphid, *Acyrthosiphon pisum*: an emerging genomic

Carlini C. R. & Grossi-de-Sa M. F., 2002. Plant toxic proteins with insecticidal properties: a review on their potentialities as bio-insecticides. *Toxicon.*, 40(11), 1515-1539. Chandra N. R., Kumar N, Jeyakani J., Singh D. D., Sharan B. Gowda S. B. & Prathima M. N., 2006. Lectindb: a plant lectin database. *Glycobiology.*, 16(10), 938-946. Chrispeels M. J. & Raikhel N. V., 1991. Lectins, lectin genes, and their role in plant defense.

Christou P., Capell T., Kohli A., Gatehouse J. A & Gatehouse A. M. R., 2006. Recent

Cole R. A., 1994. Isolation of a chitin-binding lectin with insecticidal activity in chemically-

Conner A. J., Glare T. R. & Nap J. P., 2003. The release of genetically modified crops into the environment-Part II. Overview of ecological risk assessment. *Plant J*., 33(1), 19-46. Couty A., Down R. E., Gatehouse A. M. R., Kaiser L., Pham-Delegue M.H. & Poppy G. M.,

aphid *Brevicoryne brassicae*. *Entomol. Exp. Appl*., 72, 181-187.

Aphidiidae). *J. Insect Physiol.*, 47(12), 1357-1366.

developments and future prospects in insect pest control in transgenic crops.

defined synthetic diets from two wild Brassica species with resistance to cabbage

2001. Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid, *Aphidius ervi* Haliday (Hymenoptera:

inducible alkaloid responses in wild tobacco. *Ecology.*, 71, 252-62.

Noctuidae). *J. Insect Physiol*., 45(11), 983-991.

*Forest Entomol*., 6(3), 215-222.

28, 747-755.

*Plant Cell.*, 3, 1-9.

*Trends Plant Sci*., 11, 302-308.

61(5), 1025-1033.

gut receptors of homopteran pests is correlated to its insecticidal activity.*Plant Sci*.,

Gatehouse A. M. R., 1999. The effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on *Eulophus pennicornis* (Hymenoptera: Eulophidae) a parasitoid of the tomato moth *Lacanobia oleracea* (Lepidoptera:

Gatehouse A.M. R., 2001. Effect of dietary cowpea trypsin inhibitor (CpTI) on the growth and development of the tomato moth, *Lacanobia oleracea* (Lepidoptera: Noctuidae) and on the success of the gregarious ectoparasitoid, *Eulophus*

Oral toxicity and impact on fecundity of three insecticidal proteins on the gregarious ectoparasitoid *Eulophus pennicornis* (Hymenoptera: Eulophidae). *Agric.*

Additive protective effects of incorporating two different higher plants derived

model system for ecological, developmental and evolutionary studies. *Bioessays.*,

#### **8.2 Synergistic effects on other proteins**

Sometimes the combinations effects of two or several entomotoxic proteins could be more efficient than the application of these proteins individually. For example, the insecticidal activity of protease inhibitor and α-amilase inhibitors were significantly increased when these inhibitors enzymes incorporated with lectin (Abdeen et al., 2005; Amirhusin et al., 2004; Murdock & Shade, 2002). Maqbool et al. (2001) reported that rice transgenic plant carrying three insecticidal genes including lectin gene (encoding gene GNA), cry1A and cry 2A, have enhanced levels of resistance to a wide range of different rice pests in comparison with non transgenic rice. Therefore, this approach will be one of the supplemented advantages to lectin applications in integrated pest management (IPM).

#### **9. Conclusion**

The aim of the current chapter was to present up to date information regarding effects of the lectins especially introducing the fungal lectins as natural agents to control insect pests. In recent years due to increasing the harmful effects of chemical compounds on non target organisms and our environment, a safe alternative to this approach is inevitable. Actually, lectins could be alternatives to chemical compounds for the pests control. Results from different investigations were shown that plant lectins as well as fungal lectins could be good candidates to be applied in the agriculture by biotechnologist in order to control insect pest.

#### **10. Acknowledgement**

The authors would like to acknowledge Iran National Science Foundation (INSF) for their financial support. This work was funded by a grant (No. 86025.11) from INSF.

#### **11. References**

Sometimes the combinations effects of two or several entomotoxic proteins could be more efficient than the application of these proteins individually. For example, the insecticidal activity of protease inhibitor and α-amilase inhibitors were significantly increased when these inhibitors enzymes incorporated with lectin (Abdeen et al., 2005; Amirhusin et al., 2004; Murdock & Shade, 2002). Maqbool et al. (2001) reported that rice transgenic plant carrying three insecticidal genes including lectin gene (encoding gene GNA), cry1A and cry 2A, have enhanced levels of resistance to a wide range of different rice pests in comparison with non transgenic rice. Therefore, this approach will be one of the supplemented

The aim of the current chapter was to present up to date information regarding effects of the lectins especially introducing the fungal lectins as natural agents to control insect pests. In recent years due to increasing the harmful effects of chemical compounds on non target organisms and our environment, a safe alternative to this approach is inevitable. Actually, lectins could be alternatives to chemical compounds for the pests control. Results from different investigations were shown that plant lectins as well as fungal lectins could be good candidates to be applied in the agriculture by biotechnologist in

The authors would like to acknowledge Iran National Science Foundation (INSF) for their

Abbott W. S., 1925. A method of computing the effectiveness of an insecticide.*J. Econ.*

Abdeen A., Virgos A., Olivella E., Villanueva J., Aviles X., Gabarra R & Prat S., 2005.

Allsopp P. G. & McGhie T. K., 1996. Snowdrop and wheat germ lectins as antimetabolites for the control of sugarcane white grubs. *Entomol. Exp. Appl*., 80(2), 409-414. Amirhusin B., Shade R. E., Koiwa H., Hasegawa P.M., Bressan R.A., Murdock L. L., Zhu-

Amirhusin B., Shade R. E., Koiwa H., Hasegawa P. M., Bressan R. A., Murdock L. L & Zhu-

Aronson A. I., 1994. *Bacillus thuringiensis* and its use as a biological insecticide. *In:* Janick J.,

against *Callosobruchus maculatus*. *J. insect physiol.*, 53(7), 734-740.

ed. *Plant Breeding Reviews*. New York, USA, Wiley., pp, 19-45.

Multiple insect resistance in transgenic tomato plants over- expressing two families

Salzman K., 2004. Soyacystatin N inhibits proteolysis of wheat alpha-amylase inhibitor and potentiates toxicity against cowpea weevil. *J. Econ. Entomol*., 97, 2095-

Salzman K., 2007. Protease inhibitors from several classes work synergistically

financial support. This work was funded by a grant (No. 86025.11) from INSF.

of plant proteinase inhibitors. *Plant Mol. Biol.*, 57, 189-202.

advantages to lectin applications in integrated pest management (IPM).

**8.2 Synergistic effects on other proteins**

**9. Conclusion**

order to control insect pest.

**10. Acknowledgement**

*Entomol*., 18, 265-267.

**11. References**

2100.

Lectins and Their Roles in Pest Control 221

Foissac X., Loc N.T., Christou P., Gatehouse A. M. R. & Gatehouse J. A., 2000. Resistance to

Francis F., Marty-Detraves C., Poincloux R., Baricault L., Fournier D. & Paquereau L., 2003.

Garret A., Kerlan C. & Thomas D., 1993. The intestine is a site of passage for potato leafroll

Gatehouse A. M. R., Dewey F. M., Dove J., Fenton K. A. & Pusztai A., 1984. Effect of seed

Gatehouse A. M. R., Barbieri L., Stirpe F. & Croy R. R. D., 1990. Effects of ribosome

Gatehouse A. M. R., Howe D. S., Flemming J. E., Hilder V. A., Gatehouse J. A., 1991a.

Gatehouse, J. A., Hilder, V. A. & Gatehouse, A. M. R. 1991b. Genetic engineering of plants

Gatehouse A. M. R., Shi Y., Powell K. S., Brough C., Hilder V. A., Hamilton W. D. O.,

Gatehouse A. M. R., Powell K. S., Peumans W. J., Van Damme E. J. M. & Gatehouse J. A.,

Gatehouse A. M. R., Down R. E., Powell K. S., Sauvion N., Rahbé Y., Newell C. A.,

Gatehouse A. M. R., Davison G. M., Newell C. A., Hamilton W. D. O., Burgess E. P. J.,

*maculatus*; mechanism of toxicity. *J. Sci. Food Agric*., 35(4), 373-380.

facilitates uptake of other molecules. *Eur. J. Cell Biol*., 82(10), 515-522. Francis F., Karimi J., Colinet F., Daniel Portetele D and Haubruge E., 2011. Purification of a

*Insect Physiol*., 46(4), 573-583.

Sulz. *Arch. Virol*., 131(3), 377-392.

seeds. *J. Sci. Food Agric*., 55, 63-74.

Coleoptera. *Entomol. Exp. Appl*., 54(1), 43-51.

properties. Fungal biology., 115, 1093-1099.

105-35.

342(1301), 279-286.

Francis., 35-58.

3(1), 49-63.

*Exp. Appl*., 79(3), 295-307.

green leafhopper (*Nephotettix virescens*) and brown planthopper (*Nilaparvata lugens*) in transgenic rice expressing snowdrop lectin (*Galanthus nivalis* agglutinin; GNA). *J.*

Fungal lectin, XCL, is internalized via clathrin-dependent endocytosis and

new fungal mannose-specific lectin from *Penicillium chrysogenum* and its aphicidal

virus from the gut lumen into the haemocoel in the aphid vector, *Myzus persicae*

lectins from *Phaseolus vulgaris* on the development of larvae of *Callosobruchus*

inactivating proteins on insect development–differences between Lepidoptera and

Biochemical basis of insect resistance in winged bean (*Psophocarpus tetragonolobus*)

for insect resistance. In Plant Genetic Engineering, Plant Biotechnology Series, V ol. 1, ed. D. Grierson. Blackie & Sons Ltd, London/Chapman and Hall, New York, pp.

Newell, C. A., Merryweather A., Boulter D. & Gatehouse J. A., 1993. Approaches to insect resistance using transgenic plants. *Philos. Trans. R. Soc. London. Biol. Sci.*,

1995. Insecticidal properties of plant lectins: their potential in plant protection. *In:*  Pusztai A. & Bardocz S., eds. *Lectins: biomedical perspectives*. London: Taylor &

Merryweather A. & Hamilton W. D. O., Gatehouse J. A., 1996. Transgenic potato plants with enhanced resistance to the peach-potato aphid, *Mizus persicae*. *Entomol.*

Gilbert, R. J. C. & Gatehouse J. A., 1997. Transgenic potato plants with enhanced resistance to the tomato moth, *Lacanobia oleracea*: growth room trials. *Mol. Breed*.,

Czapla T. H. & Lang B. A., 1990. Effect of plant lectins on the larval development of

Desoignies N., 2008. Étude des interactions virus-vecteur:transmission par *Myzus persicae*

Down R. E., Gatehouse A. M. R., Hamilton W. D. O. & Gatehouse J. A., 1996. Snowdrop

Down R. E., Ford L., Bedford S. J., Gatehouse L. N., Newell C., Gatehouse J. A. & Gatehouse

Elden T. C., 2000. Influence of a cysteine proteinase inhibitor on alfafa weevil (Coleptera:

Etzler M. E., 1986. Distribution and function of plant lectins. *In:* Liener I.E., Sharon N. & Goldstein I. J., eds. *The lectins*. San Diego, CA, USA: Academic Press., 371-435. Ferre J. & Rie J. V., 2002. Biochemistry and genetics of insect resistance to *Bacillus*

Ferry N., Edwards M. G., Gatehouse T., Capell P., Christou P. & Gatehouse A. M. R., 2006. Transgenic plants for insect pest control: a forward looking scientific perspective. *Transgenic*

Fitches E., Gatehouse A. M. R. & Gatehouse J. A., 1997. Effects of snowdrop lectin (GNA)

Fitches E. & Gatehouse J. A., 1998. A comparison of the short and long term effects of

Fitches E., Woodhouse S. D., Edwards J. P. & Gatehouse J. A., 2001a. *In vitro* and *in vivo*

Fitches E., Ilett C., Gatehouse A. M. R., Gatehouse L. N., Greene R., Edwards J. P. &

moth larvae (*Laconobia oleracea*). *J. Insect Physiol*., 44(12), 1213-1224.

mechanisms of insecticidal action. *J. Insect Physiol*., 47(7), 777-787.

delivered via artificial diet and transgenic plant on the development of tomato moth (*Lacanobia oleracea*) larvae in laboratory and glasshouse trials. *J. Insect Physiol*.,

insecticidal lectins on the activities of soluble and brush border enzymes of tomato

binding of snowdrop (*Galanthus nivalis* agglutinin; GNA) and jackbean (*Canavalia ensiformis*; ConA) lectins within tomato moth (*Lacanobia oleracea*) larvae;

Gatehouse J. A., 2001b. The effects of *Phaseolus vulgaris* erythro- and leucoagglutinating isolectins (PHA-E and PHA-L) delivered via artificial diet and transgenic plants on the growth and development of tomato moth (*Lacanobia oleracea*) larvae; lectin binding to gut glycoproteins *in vitro* and *in vivo*. *J. Insect*

(PLRV). Mémoire DEA : Université Catholique de Louvain (Belgique). Dimitrov D. S., 2004. Virus entry: molecular mechanisms and biomedical applications. *Nat.*

(Coleoptera: Crysomelidae). *J. Econ*. *Entomol*., 83, 2480-2485.

Dixon H. B. F., 1981. Defining a lectin. Letter to Nature. *Nature*, 292(1981), 192.

laboratory and glasshouse trial. *J. Insect Physiol*., 42(11), 1035-1045.

*Rev. Microbiol.*, 2(2), 109-122.

*Entomol.Sci*., 35, 70-76.

*Res*., 15, 13-19.

43(8), 727-739.

*Physiol*., 47(12), 1389-1398.

*thuringiensis*. *Ann. Rev. Entomol*., 47, 501-533.

236.

European corn borer (Lepidoptera: Pyralidae) and southern corn rootworm

d'un virus sur le mode non-persistant (PVY) et d'un virus sur le mode persistant

lectin inhibits development and decreases fecundity of the glasshouse potato aphid (*Aulacorthum solani*) when administered *in vitro* and via transgenic plants both in

A. M. R., 2001. Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. *Transgenic Res*., 10, 223-

Curculionidae) growth and development over successive generations. *J.*

Lectins and Their Roles in Pest Control 223

Hogenhout S. A., Ammar E. D., Whitfield A. E. & Redinbaugh M.G., 2008. Insect vector

Janmaat A. F. & Myers J., 2003. Rapid evolution and the cost of resistance to *Bacillus*

Yao J., Pang Y., Qi H., Wan B., Zhao X., Kong W., Sun X., Tang K., 2003.Transgenic Tobacco

Jongsma M. A. & Bolter C., 1997. The adaptation of insects to plant protease inhibitors. *J.*

Kanrar S., Venkateswari J., Kirti P. B. & Chopra V. L., 2002. Transgenic Indian mustard

Karimi J., Paquereau L., Fournier D., Haubruge E. & Francis F., 2006. Use of artificial diet

Karimi J., Paquereau L., Fournier D., Haubruge E. & Francis F., 2007. Effect of a fungal lectin

Karimi j., 2009. Study of fungal lectins as potential bio-pesticides to control aphid pests. Ph.D. thesis. Agricualtural University of Gembloux, Belgium. 124pp. Karimi J., Francis F., Cuartero Diaz G., Haubruge E., 2008. Investigation of Carbohydrate

Karimi J., Francis F., Haubruge E., 2010. Development of entomotoxic molecules as control

Kumar M. A., Timm D. E., Neet K. E., Owen W. G., Peumans W. J. & Rao A. G., 1993.

Lis H. & Sharon N., 1998. Lectins: carbohydrate-specific proteins that mediate cellular

an inhibitor of protein synthesis. *J. Biol. Chem*., 268(33), 25176-25183. Leple J. C., Bonade-bottino M., Agustin S., Pilate G., Dumanois Lê Tân V., Deplanque A.,

critical review. *Crop Prot*., 18(3), 177-191.

*Soc. London Ser. Biol. Sci*., 270, 2263-2270.

*Comm. Appl. Biol. Sci. Ghent Univ.*, 72(3), 629-638.

*Agro., Société et Environ., 14(1), 225-241.*

recognition. *Chem. Rev*., 98, 637-674.

*Transgenic Res*., 12(6), 715-22.

*Insect Physiol*., 43, 885- 895.

*Cell Rep*., 20, 976-981.

71(2b), 497-505.

*Sci..,* 73/3,629-938.

*Mol. Breed*., 1, 319-328.

359.

Van Damme E.J.M. & Boulter D., 1995. Expression of snowdrop lectin in transgenic tobacco results in added protection against aphids. *Transgenic Res*., 4(1), 18-25. Hilder V.A. & Boulter D., 1999. Genetic engineering of crop plants for insect resistance: a

interactions with persistently transmitted viruses. *Ann. Rev. Phytopathol*., 46, 327-

*thuringiensis* in greenhouse populations of cabbage loopers, *Trichoplusia ni*. *Proc. R.*

Expressing *Pinellia ternata* Agglutinin Confers Enhanced Resistance to Aphids.

(*Brassica juncea*) with resistance to the mustard aphid (*Lipaphis erysimi* Kalt.). *Plant*

system to assess the potential bio-insecticide effect of a fungal lectin from *Xerocomus chrysenteron* (XCL) on *Myzus persicae*. *Comm. Appl. Biol. Sci. Ghent Univ.*,

from *Xerocomus chrysenteron* (XCL) on the biological parameters of *Myzus persicae*.

binding property of a fungal lectin from *Xerocomus chrysenteron* and potential use on *Myzus persicae* aphid. *Ghent University* (in press). *Comm. in Agricul. and Appl. Biol.*

agents: illustration of some protein potential uses and limits of lectins. *Biotech.,*

Characterization of the lectin from the bulbs of *Eranthis hyemalis* (winter aconite) as

Coru D. & Jouanin L., 1995. Toxicity to *Chrysomela tremulae* (Coleoptera: Crysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor.

Gatehouse A. M. R. & Gatehouse J. A., 1998. Identifying proteins with insecticidal activity:

Gatehouse A. M. R, Davison G. M., Stewart J. N., Galehouse L. N., Kumar A., Geoghegan I.

Gildow F. E., 1993. The aphid salivary gland basal lamina as a selective barrier associated

Goldstein I. J. & Poretz R. D., 1986. Isolation, physicochemical characterization, and

Gray S. M., 1996. Plant virus proteins involved in natural vector transmission. *Trends*

Gray S. M. & Banerjee N., 1999. Mechanisms of arthropod transmission of plant and animal

Gray S. M. & Gildow F. E., 2003. Luteovirus-aphid interactions. *Annu. Rev. Phytopathol*., 41,

Gurjar M. M., Gaikwad S. M., Haila Salokhe S. G, Mukherjee S & Islamkhan M., 2000.

Habibi J. E., Backus E. A. & Czapla T. M., 1993. Plant lectins affect survival of the potato leaf-

Habibi J. E., Backus E. A. & Czapla T. H., 1998. Subcellular effects and localization of

Habibi J. E., Backus E. A. & Huesing J. E., 2000. Effects of phytohemagglutinin (PHA) on the

tarnished plant bug *Lygus hesperus* Knight. *J. Insect Physiol*., 46(5), 611-619. Halitschke R., Schittko U., Pohnert G., Boland W. & Baldwin I.T., 2001. Molecular

Hamshou M, Smagghe G., Shahidi-Noghabi S., De Geyter E., Lannoo N., Van Damme EJM.,

Hamshou M., Smagghe G., Van Damme E. J. M., 2010. Entomotoxic effects of fungal lectin fromRhizoctonia solani towards Spodoptera littoralis. *Fungal Biol*., 114, 34-40. Hilder V. A., Gatehouse A. M. R., Sherman S. E., Baler R. F. & Boulter D., 1987. A novel mechanism for insect resistance engineered into tobacco. *Nature.*, 330, 160-163. Hilder V. A., Powell K. S., Gatehouse A. M. R., Gatehouse J. A., Gatehouse L. N., Shi Y.,

hopper (Homoptera: Cicadellidae). *J. Econ. Entomol*., 86(3), 945-951.

(Insecta: Homoptera: Cicadellidae). *Cell Tissue Res*., 294(3), 561-571.

herbivorespecific plant responses. *Plant Physiol*., 125(4), 711-717.

Growth inhibition and total loss of reproductive potential in *Tribolium castaneum* by

binding sites of phytohemagglutinin in the potato leafhopper, *Empoasca fabae*

structure of midgut epithelial cells and localization of its binding sites in western

interactions between the specialist herbivore *Manduca sexta* (Lepidoptera, Sphingidae) and its natural host *Nicotiana attenuata*. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for

2010. Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells. *Insect Biochem. and Molecul. Biol.,*40, 883-890.

Hamilton W. D. O., Merryweather A., Newell C. A., Timans J. C., Peumans W. J.,

expressed in transgenic potato plants. *Mol. Breed*., 5(2), 153-165.

J., eds. *The lectins*. Orlando, FL, USA: Academic Press., 33-247.

viruses. *Microbiol. Mol. Biol. Rev*., 63(1), 128-148.

*Artocarpus hirsut* lectin. *Invertebr. Reprod. Dev*., 38, 95-98.

*Phytopathology.*, 83(12), 1293-1302.

*Microbiol*., 4(7), 259-264.

165-175.

539-566.

use of encoding genes to produce insect-resistant transgenic crops. *Pest Sci.*, 52(2),

E., Birch A. N. E. & Gatehouse J. A., 1999. Concanavalin A inhibits development of tomato moth (*Lacanobia oleracea*) and peach-potato aphid (*Myzus persicae*) when

with vector-specific transmission of barley yellow dwarf luteoviruses.

carbohydrate-binding specificity of lectins. *In:* Liener I.E., Sharon N. & Goldstein I.

Van Damme E.J.M. & Boulter D., 1995. Expression of snowdrop lectin in transgenic tobacco results in added protection against aphids. *Transgenic Res*., 4(1), 18-25.

Lectins and Their Roles in Pest Control 225

Ng T. B., Chan W. Y. & Yeung H. W., 1992. Proteins with abortifacient, ribosome

Okeola O. G. & Machuka J., 2001. Biological effects of African yam bean lectins on *Clavigralla*

Peumans W. J. & Van Damme E. J. M., 1995. Lectins as plant defense proteins. *Plant Physiol*.,

Peumans W. J., Hao Q. & Van Damme E. J. M., 2001. Ribosome-inactivating proteins from

Powell K. S., 2001. Antimetabolic effects of plant lectins towards nymphal stages of the

Powell K. S., Gatehouse A. M. R., Hilder V. A. & Gatehouse J. A., 1993. Antimetabolic effects

Powell K. S., Gatehouse A. M. R., Hilder V. A., Van Damme E. J. M., Peumans W. J.,

Powell K. S., Spence J., Bharathi M., Gatehouse J. A. & Gatehouse A. M. R., 1998.

Rahbé Y. & Febvay G., 1993. Protein toxicity to aphid: an *in vitro* test on *Acyrthosiphon pisum*.

Rahbe´ Y., Sauvion N., Febvay G., Peumans W. J. & Gatehouse A. M. R., 1995. Toxicity of

Rahbé Y., Deraison C., Bonadé-Bottino M., Girard C., Nardon C., Jouanin L., 2003. Effects of

Rahbé Y., Ferrasson E., Rabesona H. & Quillien L., 2003b. Toxicity to the pea aphid

protease inhibitors from pea seeds. *Insect Biochem. Mol. Biol*., 33, 299-306. Ranjekar P. K., Patankar A., Gupta V., Bhatnagar R., Bentur J. & Kumar P. A., 2003. Genetic engineering of crop plants for insect resistance. *Curr. Sci*., 84, 321-329. Rao K.V., Rathore K. S., Hodges T. K., Fu X., Stoger E., Sudhakar D., Williams S., Christou

plants confers resistance to rice brown planthopper. *Plant J*., 15(4), 469-477. Roy A., Banerjee S., Majumder P. & Das S., 2002. Efficiency of mannose-binding plant lectins

planthoppers *Tarophagous proserpina* and *Nilaparvata lugens*. *Entomol. Exp. Appl*.,

of plants lectins and fungal enzymes on the nymphal stages of two important rice pests, *Nilaparvata lugens* and *Nephotettix cinciteps*. *Entomol. Exp. Appl*., 66(2), 119-126.

Boonjawat J., Horsham K. & Gatehouse J. A., 1995. Different antimetabolic effects of related lectins towards nymphal stages of *Nilaparvata lugens*. *Entomol. Exp. Appl*.,

Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper *Nilaparvata lugens*

lectins and processing of ingested proteins in the pea aphid *Acyrthosiphon pisum*.

the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of *Myzus persicae* on OC-I expression transgenic oilseed rape.

*Acyrthosiphon pisum* of antichymotrypsin isoforms and fragments of Bowman-Birk

P., Bharathi M., Bown D. P., Powell K. S., Spence J., Gatehouse A. M. R. & Gatehouse J. A., 1998. Expression of snowdrop lectin (GNA) in transgenic rice

in controlling a homopteran insect, the red cotton bug. *J. Agric. Food Chem*., 50,

*tomentosicollis* (Hemiptera: Coreidae). *J. Econ. Entomol*., 94, 724-729.

plants: more than RNA N-glycosidases. *FASEB J*., 15(9), 1493-1506.

Cucurbitaceae plants. *Gen. Pharmacol*., 23(4), 575-590.

109(2), 347-352.

99(1), 71-77.

75(1), 61-65.

(Stal). *J. Insect Physiol*., 44(7), 529-539.

*Entomol. Exp. Appl*., 67(2), 149-160.

*Entomol. Exp. Appl.*, 76, 143-155.

*Plant Sci.*, 164(4), 441-450.

6775-6779.

inactivating, immunomodulatory, antitumor and anti-AIDS activities from

Loc N. T., Tinjuangjun P., Gatehouse A. M. R., Christou P. & Gatehouse J. A., 2002. Linear

Ma Q. H., Tian B., Li Y. L., 2010. Overexpression of a wheat jasmonate-regulated lectin

Macedo M. L. R., Freire M. G. M., Novello J. C. & Marangoni S., 2002. *Talisia esculenta* lectin

Macedo M. L. R., Damico D. C., Freire M. G. M., Toyama M. H., Marangoni S. & Novello

Macedo M. L. R., Freire M. G. M., Silva M. B. R. & Coelho L.C. B. B., 2006. Insecticidal action

Machuka J., Van Damme E. J. M., Peumans W. J. & Jackai L. E. N., 1999. Effect of plant

Machuka J. S., Okeola O. G., Chrispeels M. J. & Jackai L. E. N., 2000. The African yam bean

development of larvae of the legume pod borer. *Phytochemistry.*, 53, 667-674. Maddock S. E., Hufman G., Isenhour D. J., Roth B. A., Raikhel N. V., Howard J. A. & Czapla

Majumder P., Mondal H. A. & Das S., 2005. Insecticidal activity of *Arum maculatum* tuber

Maqbool S. B., Riazuddin S., Loc N. T., Gatehouse A. M. R., Gatehouse J. A. & Christou P.,

McGaughey W. H. & Whalon M. E., 1992. Managing insect resistance to *Bacillus thuringiensis*

Murdock L. L. & Shade R. E., 2002. Lectins and protease inhibitors as plant defense against

Naidu R. A., Ingle C. J., Deom C. M. & Sherwood J. L., 2004. The two envelope membrane

Nelson D. & Cox M., 2005. *Lehninger principles of biochemistry*. 4th ed. Freeman. Ng T.B., 2004.

properties and sensitivities to glycosidases. *Virology.*, 319, 107-117.

Peptides and proteins from fungi. *Peptides.*, 25(6), 1055-1073.

against a range of different rice pest. *Mol. Breed*., 9, 231-244.

(Coleoptera: Bruchidae). *Biochim. Biophys.Acta.*, 1571, 83-88.

(Lepidoptera: Pyralidae). *J. Agric. Food Chem*., 51, 2980-2986.

Bruchidae). *Comp. Biochem. Physiol*., 146, 486-498.

range of different rice pests. *Mol. Breed*., 7, 85-93.

insects. *J. Agric. Food Chem*., 50(22), 6605-6611.

Marsh M. & Helenius A., 2006. Virus entry: open sesame. *Cell*, 124(4), 729-740.

93, 179-187.

*Arizona, USA*.

53(17), 6725-6729.

toxins. *Science.*, 258, 1451-1455.

increases pathogen resistance. *Biochimie.,* 92, 187–193.

transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

and larval development of *Callosobruchus maculatus* and *Zabrotes subfasciatus*

J.C., 2003. Purification and characterization of an N-acetylglucosamine-binding lectin from *Koelreuteria paniculata* seeds and its effect on the larval development of *Callosobruchus maculatus* (Coleoptera: Bruchidae) and *Anagasta kuehniella*

of *Bauhinia monandra* leaf lectin (BmoLL) against *Anagasta kuehniella* (Lepidoptera: Pyralidae), *Zabrotes subfasciatus* and *Callosobruchus maculatus* (Coleoptera:

lectins on survival development of the pod borer *Maruca vitrata*. *Entomol.Exp. Appl*.,

seed lectin affects the development of the cowpea weevil but does not affect the

T. H., 1991. Expression in maize plants of wheat germ agglutinin, a novel source of insect resistance. *In: Third International Congress in Plant Molecular Biology, Tucson,*

lectin and its binding to the glycosylated insect gut receptors. *J. Agric. Food Chem*.,

2001. Expression of multiple insecticidal genes confers broad resistance against a

glycoproteins of tomato spotted wilt virus show differences in lectin-binding

Lectins and Their Roles in Pest Control 227

Thielens N. M., Tacnet-Delorme E. P. & Arlaud G. J., 2002. Interaction of C1q and mannan-

Tinjuangjun P., Loc N. T., Gatehouse A. M. R., Gatehouse J. A. & Christou P., 2000.

Tomov B. W. & Bernal J. S., 2003. Effects of GNA transgenic sugarcane on life history

Trigueros V., Lougarre A., Ali-Ahmed D., Rahbe Y., Guillot J., Chavant L., Fournier D. &

Van Damme E. J. M., Peumans W. J., Pusztai A. & Bardocz S., 1998. *Handbook of plant lectins: properties and biomedical applications*. Bognor Regis, UK: John Wiley and Sons. Vasconcelos I. M. & Oliveira J. T. A., 2004. Antinutritional properties of plant lectins.

Wang G., Zhang J., Song F., Wu J., Feng S. & Huang D., 2006. Engineered *Bacillus*

Wang H., Ng T. B. & Liu Q., 2003. A novel lectin from the wild mushroom *Polyporus adusta*.

Wang H. X, Ng T. B. & Ooi V. E. C., 1998. Lectins from mushroom. *Mycol. Res*., 102, 897-906. Wang M., Trigueros V., Paquereau L., Chavant L. & Fournier D., 2002. Proteins as active

Wang Z. B. & Guo S. D., 1999. Expression of two insect resistant genes cryIA (bandc) GNA

Wei T., Chen H., Ichiki-Uehara T., Hibino H. & Omura T., 2007. Entry of rice dwarf virus

Wu A., Sun X., Pang Y. & Tang K., 2002. Homozygous transgenic rice lines expressing GNA

Yamada T., Hattori K. & Ishimoto M., 2001. Purification and characterization of two �-

Zhao J. Z., Cao J., Li Y. X., Collins H. L., Roush R.T., Earle E. D., Shelton A. M., 2003.

Zhu K., Joseph E. H., Richard E. S., Ray A. B., Paul M. H., Larry L. M., 1996. An insecticidal

Zhu-Salzman K., Shade R.E., Koiwa H., Salzman R.A., Narasimhan M., Bressan, R.A.,

Enhanced insect resistance in Thai rice varieties generated by particle

parameters of *Parallorhogas pyralophagus* (Marsh) (Hymenoptera: Braconidae), a

Paquereau L., 2003. *Xerocomus chrysenteron* lectin: identification of a new pesticidal

*thuringiensis* G033A with broad insecticidal activity against lepidopteran and

compounds involved in insecticidal activity of mushroom fruitbodies. *J.*

in transgenic tobacco plants results in added protection against both cotton

into cultured cells of its insect vector involves clathrin-mediated endocytosis. *J.*

with enhanced resistance to the rice sap-sucking pest *Laodelphax striatellus*. *Plant*

amylase inhibitors from seeds of tepary bean (*Phaseolus acutifolius* A.gray).

Transgenic plants expressing two *Bacillus thuringiensis* toxins delay insect resistance

N-acetylglucosamine specific lectin gene from *Griffonia simplicifolia* (Leguminosae).

Hasegawa P.M. & Murdock L.L., 1998. Carbohydrate binding and resistance to

binding lectin with viruses. *Immunobiology.*, 205, 563-574.

parasitoid of Mexican rice borer. *J. Econ. Entomol*., 96, 570-576.

coleopteran pests*. Appl. Microbiol.Biotechnol*., 72(5), 924-930.

bombardment. *Mol. Breed*., 6, 391-399.

protein. *Entomol. Exp. Appl*., 1621(3), 292-298.

*Biochem. Biophys. Res. Commun*., 307, 535-539.

bollworm and aphids. *Chin*. *Sci. Bull*., 44, 2051-2058.

*Toxicon.*, 44(4), 385-403.

*Econ.Entomol*., 95, 603-617.

*Virol*., 81(14), 7811-7815.

*Phytochemistry.*, 58(1), 59-66.

*Plant Physiol*., 110, 195-202.

evolution. *Nat. Biotechnology*., 21, 1493-1497.

*Breed*., 121, 93-95.

Rudiger H. & Gabius H. J., 2001. Plant lectins: occurrence, biochemistry, functions and

Sauvion N., Rahbe Y., Peumans W. J., Van Damme E. J. M., Gatehouse J. A. & Gatehouse A.

Sauvion N., Nardon C., Febvay G., Angharad M., Gatehouse R. & Rahbe Y., 2004. Binding

Schnepf H. E. & Whiteley H. R., 1981. Cloning and expression of the *Bacillus thuringiensis*  crystal protein gene in *Escherichia coli*. *Proc. Natl Acad. Sci. USA*, 78(5), 2893-2897. Schuler T. H., Poppy G. M., Kerry B. R. & Denholm I., 1998. Insect resistant transgenic

Scott J. G. & Wen Z. M., 2001. Cytochromes P450 of insects: the tip of the iceberg. *Pest*

Setamou M., Bernal J. S., Legaspi J. C., Mirkov T. E. & Legaspi B. C., 2002. Evaluation of

Shahidi-Noghabi S., Van Damme E. J. M. & Smagghe G., 2008. Carbohydrate-binding

Shahidi-Noghabi S., Van Damme E. J. M. & Smagghe G., 2009. Expression of *Sambucus nigra*

enhanced resistance to different insect species. *Transgenic Res*., 18, 249-259. Smith C. M. & Boyko L. V., 2006. The molecular bases of plant resistance and defense responses to aphid feeding: current status. *Entomol. Exp. Appl*., 122(1), 1-16. Stillmark H., 1888. Ueber Ricin, ein giftiges Ferment aus dem Samen von *Ricinus communis*  L. und einigen anderen Euphorbiaceen. *Arb. Pharmak. Inst. Dorpat*., 3, 59-151. Stirpe F. & Battelli M.G., 2006. Ribosome-inactivating proteins: progress and problems. *Cell*

Stoger E., Williams S., Christou P., Down R. E. & Gatehouse J. A., 1999. Expression of the

Sun X., Wu A. & Tang K., 2002. Transgenic rice lines with enhanced resistance to the small

Tabashnik B. E., Cushing N. L., Finson N. & Johnson M. W., 1990. Field development of

Tang K., Zhao E., Sun X., Wan B., Qi H. & Lu X., 2001. Production of transgenic rice

insecticidal lectin from snowdrop (*Galanthus nivalis* agglutinin, GNA) in transgenic wheat plants: effects predation by the grain aphids *Sitobion avenae*. *Mol. Breed*., 5(1),

resistance to *Bacillus thuringiensis* in diamondback moth (Lepidoptera: Plutellidae).

homozygous lines with enhanced resistance to the rice brown planthopper. *Acta.*

effects on life history parameters. *J. Econ. Entomol*., 95(2), 469-477.

lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae):

activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (*Sambucus nigra*) is a determining factor for its insecticidal activity. *Phytochemistry.*,

agglutinin (SNAI0) from elderberry bark in transgenic tobacco plants results in

M. R., 1996. Effects of GNA and other mannose binding lectins on development and fecundity of the potato-peach aphid *Myzus persicae*. *Entomol. Exp.Appl*., 79, 285-

of the insecticidal lectin Concanavalin A in pea aphid, *Acyrthosiphon pisum* (Harris) and induced effects on the structure of midgut epithelial cells. *J. Insect Physiol*.,

applications. *Glycoconjugate J*., 18(8), 589-613.

plants. *Trends Biotechnol*., 16, 168-175.

*Manage. Sci*., 57(10), 958-967.

*Mol. Life Sci.*, 63(16), 1850-1866.

*J. Econ. Entomol*., 83, 1671-1676.

*Biotechnol*., 21, 117-128.

brown planthopper. *Crop Prot*., 21(6), 511-514.

293.

50(12), 1137-1150.

69(17), 2972-2978.

65-73.

**11**

*1Iran 2Brazil*

**Plant Proteinaceous α-Amylase and Proteinase**

**Inhibitors and Their Use in Insect Pest Control**

During the evolution, plants have developed strategies to maintain favorable growth and also guarantee their survival. Enhancing the protective mechanisms, for example, is one of these strategies that allow them to successfully tolerate/resist insects, phytopathogenic microorganisms, and other unfavorable conditions (Jackson and Tailor, 1996; Malek and Dietrich, 1999; Stotz et al, 1999). Proteinaceous molecules such as α-amylase inhibitors (α-AI) and proteinase inhibitors (PIs), lectins, some hydrolyzing enzymes (e.g. b-1,3 glucanases and chitinases), and also antimicrobial peptides, are important part of protective mechanisms in plants (Fritig et al., 1998; Howe, 2008). Host plant resistance and natural plant products offer a potentially benign method for insect pest control. They are safe to the non-target beneficial organisms and human beings (Andow, 2008). This kind of plant resistance can be utilized as an economic means to reduce crop losses arising from insect pest. The wild accessions of wheat and barley do not have effective resistance and therefore plant traits that contribute to pest resistance need to be reinforced using new approaches. Thus, using the new control methods are needed such as α-amylase inhibitors, protease inhibitors, lectins and possibly δ-endototoxin (Sharma and Ortiz,

In recent years, attentions have been focused on the idea of using digestive enzyme inhibitors that affect the growth and development of pest species (Mehrabadi et al., 2010, 2011, 2012). Inhibitors of insect α-amylase, proteinase and other plant proteins have already been demonstrated to be an important biological system in the control of insect pests (Chrispeels *et al*., 1998; Gatehouse and Gatehouse, 1998; Morton et al., 2000; Valencia *et al*., 2000; Carlini and Grossi-de-Sa´, 2002; Svensson *et al*., 2004; Barbosa et al., 2010). Different types of proteinaceous α-AIs are found in microorganisms, plants and animals. Cereals such as wheat, barley, rye, rice and sorghum contain small amylase inhibitors about 18 kDa in size (Abe *et al*., 1993; Feng *et al*., 1996; Yamagata *et al*., 1998; Iulek *et al*., 2000). There are different kinds of inhibitors that potentially are a good source of α-amylases inhibitors that could be used against insect pest (Franco *et al*. 2002; Svensson et al., 2004). They show

**1. Introduction**

2000) to diminish reliance on insecticides.

Mohammad Mehrabadi1, Octavio L. Franco2 and Ali R. Bandani1

*1Department of Plant Protection, University of Tehran, Karaj, 2Centro de Análise Proteômicas e Bioquímicas de Brasília,*

*Universidade Católica de Brasília, Brasília-DF,*

proteolysis control insecticidal activity of *Griffonia simplicifolia* lectin II. *Proc. Natl Acad. Sci.USA.*, 95, 15123-15128.

Zhu-Salzman K. & Salzman R., 2001. A functional mechanic of the plant defensive *Griffonia simplicifolia* lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut. *J. Econ. Entomol*., 94, 1280-1284.

### **Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control**

Mohammad Mehrabadi1, Octavio L. Franco2 and Ali R. Bandani1 *1Department of Plant Protection, University of Tehran, Karaj, 2Centro de Análise Proteômicas e Bioquímicas de Brasília, Universidade Católica de Brasília, Brasília-DF, 1Iran*

*2Brazil*

#### **1. Introduction**

228 New Perspectives in Plant Protection

Zhu-Salzman K. & Salzman R., 2001. A functional mechanic of the plant defensive *Griffonia*

binding in the insect gut. *J. Econ. Entomol*., 94, 1280-1284.

*Acad. Sci.USA.*, 95, 15123-15128.

proteolysis control insecticidal activity of *Griffonia simplicifolia* lectin II. *Proc. Natl*

*simplicifolia* lectin II: resistance to proteolysis is independent of glycoconjugate

During the evolution, plants have developed strategies to maintain favorable growth and also guarantee their survival. Enhancing the protective mechanisms, for example, is one of these strategies that allow them to successfully tolerate/resist insects, phytopathogenic microorganisms, and other unfavorable conditions (Jackson and Tailor, 1996; Malek and Dietrich, 1999; Stotz et al, 1999). Proteinaceous molecules such as α-amylase inhibitors (α-AI) and proteinase inhibitors (PIs), lectins, some hydrolyzing enzymes (e.g. b-1,3 glucanases and chitinases), and also antimicrobial peptides, are important part of protective mechanisms in plants (Fritig et al., 1998; Howe, 2008). Host plant resistance and natural plant products offer a potentially benign method for insect pest control. They are safe to the non-target beneficial organisms and human beings (Andow, 2008). This kind of plant resistance can be utilized as an economic means to reduce crop losses arising from insect pest. The wild accessions of wheat and barley do not have effective resistance and therefore plant traits that contribute to pest resistance need to be reinforced using new approaches. Thus, using the new control methods are needed such as α-amylase inhibitors, protease inhibitors, lectins and possibly δ-endototoxin (Sharma and Ortiz, 2000) to diminish reliance on insecticides.

In recent years, attentions have been focused on the idea of using digestive enzyme inhibitors that affect the growth and development of pest species (Mehrabadi et al., 2010, 2011, 2012). Inhibitors of insect α-amylase, proteinase and other plant proteins have already been demonstrated to be an important biological system in the control of insect pests (Chrispeels *et al*., 1998; Gatehouse and Gatehouse, 1998; Morton et al., 2000; Valencia *et al*., 2000; Carlini and Grossi-de-Sa´, 2002; Svensson *et al*., 2004; Barbosa et al., 2010). Different types of proteinaceous α-AIs are found in microorganisms, plants and animals. Cereals such as wheat, barley, rye, rice and sorghum contain small amylase inhibitors about 18 kDa in size (Abe *et al*., 1993; Feng *et al*., 1996; Yamagata *et al*., 1998; Iulek *et al*., 2000). There are different kinds of inhibitors that potentially are a good source of α-amylases inhibitors that could be used against insect pest (Franco *et al*. 2002; Svensson et al., 2004). They show

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control 231

amylase. The three-dimensional model of TMA consists of a single polypeptide chain of 471 amino acid residues, one calcium ion, one chloride ion and 261 water molecules. The enzyme consists of three distinct domains, A (residues 1 to 97 and 160 to 379), B (residues 98 to 159) and C (residues 380 to 471) (Strobl et al, 1998). A representation of the overall polypeptide folds, as well as the location of the bound ions and the residues presumably involved in catalysis. Domain A is the central domain with (β / α)8-barrels comprises the core of the molecule and also includes the catalytic residues (Asp 185, Glu 222 and Asp 287). Two other domains (Domains B and C) are opposite each other, on each side of domain A. The Ca2+ binding site in TMA is located at the interface of the domain A central β-barrel and domain B. This ion is important for the structural integrity of TMA. TMA has also chloride-binding site on the same side of the β-barrel as the catalytic and the calcium-binding site, in the vicinity of both. Insect α-amylases are closely related to mammalian α-amylases (Strobl et al., 1997). The most striking difference between mammalian and insect α-amylases is the presence of additional loops in the vicinity of the active site of the mammalian enzymes (Strobl et al., 1998a). α-Amylases are the most important digestive enzymes of many insects that feed exclusively on seed products during larval and/or adult life. When the action of the amylases is inhibited, nutrition of

α-AIs are abundant in microorganisms, higher plants, and animals (Da Silva et al., 2000; Toledo et al., 2007). These organisms produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. α-Amylase inhibitors can be extracted from several plant species including legumes (Marshall and Lauda, 1975; Ishimoto et al., 1996; Grossi-de-Sa et al., 1997) and cereals (Abe et al., 1993; Feng et al., 1996; Yamagata et al., 1998; Franco et al., 2000; Iulek et al., 2000). Diverse α-amylase inhibitors reveal different characteristics against various α-amylases (Franco et al., 2002). In Table 1 inhibitory activity of αAIs from different sources are reviewed. α-AIs are naturally used by plants as a defense mechanism against insect pests (Ishimoto et al., 1989; Kluh et al., 2005). Moreover, there is a great interest to use α-AIs for control of insect pest and also to use them for production of transgenic plants that are resistant against insect pest (Gatehouse et al., 1998; Chrispeels et al., 1998; Gatehouse and Gatehouse, 1998; Valencia et al., 2000; Yamada et al., 2001). Inhibitors to insect α-amylase have already been demonstrated to be an important biological system in the control of insect pests (Franco et al., 2002; Carlini and Grossi-de-Sa´, 2002; Svensson *et al*., 2004). The expression of α-amylase inhibitors has been showed to be effective in transgenic plants. The expression of the cDNA encoding αAl-I into some plants such as pea (*Pisum sativum* L.) and azuki bean (*Vigna anguralis* L.) against bruchid beetle pests (Coleoptera: Bruchidae) has been well documented for showing ability of these inhibitors to be used as plant resistance factors against some species of insect pests (Ishimoto et al., 1989; Yamada et al., 2001; Kluh et al., 2005). Pea and azuki transgenic plants expressing α-amylase inhibitors from common beans (α-AI) were completely resistant to the *Bruchus pisorum* and *Callosobruchus chinensis* weevils (Morton *et al.* 2000). Rye α-amylase inhibitor expressed in transgenic tobacco seeds (*Nicotiana tabacum*) caused 74% mortality in *Anthonomus grandis* first instar larvae when transgenic seed flour mixture used in artificial

the organism is impaired causing shortness in energy.

diet (Dias et al., 2010).

**3. Proteinaceous α-amylase inhibitors from plants**

diverse structural differences thus causing different mode of actions and diverse specificity against target enzymes. Different α-amylase inhibitors have different modes of action against α-amylases for example inhibitors extracted from cereals and beans (*Phaseolus vulgaris*) have different molecular structures, leading to different modes of inhibition and different specificity against a diverse range of α-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own αamylases or human amylases and must not change the nutritional value of the crop (Franco *et al*. 2002; Svensson et al., 2004).

Proteinase inhibitors are capable of interfering with insect protein digestion by binding to digestive proteases of phytophagous insects, resulting in an amino acid deficiency thus affecting insect growth and development, fecundity, and survival (Lawrence and Koundal 2002; Oppert et al. 2003; Azzouz et al. 2005). They along with α-amylases inhibitors constitute major tools for improving the resistance of plants to insects. Transgenic plants expressing serine and systeine proteinase inhibitors have shown resistance to some insect pest species including Lepidoptera and Coleoptera (De Leo et al. 2001; Falco and Silva-Filho 2003; Alfonso-Rubi et al. 2003). PIs are the products of single genes, therefore they have practical advantages over genes encoding for complex pathways and they are effective against a wide range of insect pests, i.e. transferring trypsin inhibitor gene from *Vigna unguiculata* to tobacco conferred resistance against lepidopteran insect species such as *Heliothis* and *Spodoptera*, and coleopteran species such as *Diabrotica* and *Anthonomus* (Hilder et al. 1987).

#### **2. Insect α-amylases**

α-Amylases (α -1,4-glucan-4-glucanohydrolases; EC <IP_ADDRESS>) are one of the most widely enzyme complexes encountered in animals, higher and lower plants, and microbes. Because of their importance in organism growth and development, these enzymes from different origins including bacteria, nematodes, mammals and insects have been purified and their physical and chemical properties characterized (Baker, 1991; Nagaraju and Abraham, 1995; Zoltowska, 2001; Rao et al., 2002; Valencia et al., 2000; Mendiola-Olaya et al., 2000; Oliveira-Nato et al., 2003 Mohammed, 2004; Bandani et al, 2009; Mehrabadi et al, 2009). Many phytophagus insects, like stored product insects, live on a polysaccharide-rich diet and are dependent on their α-amylases for survival (Mendola-Olaya et al. 2000; Boyd et al. 2002; Mehrabadi et al, 2011). They convert starch to maltose, which is then hydrolyzed to glucose by α-glucosidase. In insects, only α-amylases that hydrolyse α-1,4- glucan chains such as starch or glycogen have been found (Terra et al. 1999).

According to Terra and Ferreira (1994), insect α-amylases generally have molecular weights in the range 48–60 kDa, pI values of 3.5–4.0, and Km values with soluble starch around 0.1%. pH optima generally correspond to the pH prevailing in midguts from which the amylases were isolated. Insect amylases are calcium-dependent enzymes, and are activated by chloride with displacement of the pH optimum. Activation also occurs with anions other than chloride, such as bromide and nitrate, and it seems to depend upon the ionic size ( Terra and Ferreira, 1994).

Although the sequences of several insect α-amylases are known, the best characterized insect α-amylase whose 3D-structure has been resolved is *Tenebrio molitor* (TMA) αamylase. The three-dimensional model of TMA consists of a single polypeptide chain of 471 amino acid residues, one calcium ion, one chloride ion and 261 water molecules. The enzyme consists of three distinct domains, A (residues 1 to 97 and 160 to 379), B (residues 98 to 159) and C (residues 380 to 471) (Strobl et al, 1998). A representation of the overall polypeptide folds, as well as the location of the bound ions and the residues presumably involved in catalysis. Domain A is the central domain with (β / α)8-barrels comprises the core of the molecule and also includes the catalytic residues (Asp 185, Glu 222 and Asp 287). Two other domains (Domains B and C) are opposite each other, on each side of domain A. The Ca2+ binding site in TMA is located at the interface of the domain A central β-barrel and domain B. This ion is important for the structural integrity of TMA. TMA has also chloride-binding site on the same side of the β-barrel as the catalytic and the calcium-binding site, in the vicinity of both. Insect α-amylases are closely related to mammalian α-amylases (Strobl et al., 1997). The most striking difference between mammalian and insect α-amylases is the presence of additional loops in the vicinity of the active site of the mammalian enzymes (Strobl et al., 1998a). α-Amylases are the most important digestive enzymes of many insects that feed exclusively on seed products during larval and/or adult life. When the action of the amylases is inhibited, nutrition of the organism is impaired causing shortness in energy.

#### **3. Proteinaceous α-amylase inhibitors from plants**

230 New Perspectives in Plant Protection

diverse structural differences thus causing different mode of actions and diverse specificity against target enzymes. Different α-amylase inhibitors have different modes of action against α-amylases for example inhibitors extracted from cereals and beans (*Phaseolus vulgaris*) have different molecular structures, leading to different modes of inhibition and different specificity against a diverse range of α-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own αamylases or human amylases and must not change the nutritional value of the crop

Proteinase inhibitors are capable of interfering with insect protein digestion by binding to digestive proteases of phytophagous insects, resulting in an amino acid deficiency thus affecting insect growth and development, fecundity, and survival (Lawrence and Koundal 2002; Oppert et al. 2003; Azzouz et al. 2005). They along with α-amylases inhibitors constitute major tools for improving the resistance of plants to insects. Transgenic plants expressing serine and systeine proteinase inhibitors have shown resistance to some insect pest species including Lepidoptera and Coleoptera (De Leo et al. 2001; Falco and Silva-Filho 2003; Alfonso-Rubi et al. 2003). PIs are the products of single genes, therefore they have practical advantages over genes encoding for complex pathways and they are effective against a wide range of insect pests, i.e. transferring trypsin inhibitor gene from *Vigna unguiculata* to tobacco conferred resistance against lepidopteran insect species such as *Heliothis* and *Spodoptera*, and coleopteran species such as *Diabrotica* and *Anthonomus*

α-Amylases (α -1,4-glucan-4-glucanohydrolases; EC <IP_ADDRESS>) are one of the most widely enzyme complexes encountered in animals, higher and lower plants, and microbes. Because of their importance in organism growth and development, these enzymes from different origins including bacteria, nematodes, mammals and insects have been purified and their physical and chemical properties characterized (Baker, 1991; Nagaraju and Abraham, 1995; Zoltowska, 2001; Rao et al., 2002; Valencia et al., 2000; Mendiola-Olaya et al., 2000; Oliveira-Nato et al., 2003 Mohammed, 2004; Bandani et al, 2009; Mehrabadi et al, 2009). Many phytophagus insects, like stored product insects, live on a polysaccharide-rich diet and are dependent on their α-amylases for survival (Mendola-Olaya et al. 2000; Boyd et al. 2002; Mehrabadi et al, 2011). They convert starch to maltose, which is then hydrolyzed to glucose by α-glucosidase. In insects, only α-amylases that hydrolyse α-1,4- glucan chains such

According to Terra and Ferreira (1994), insect α-amylases generally have molecular weights in the range 48–60 kDa, pI values of 3.5–4.0, and Km values with soluble starch around 0.1%. pH optima generally correspond to the pH prevailing in midguts from which the amylases were isolated. Insect amylases are calcium-dependent enzymes, and are activated by chloride with displacement of the pH optimum. Activation also occurs with anions other than chloride, such as bromide and nitrate, and it seems to depend

Although the sequences of several insect α-amylases are known, the best characterized insect α-amylase whose 3D-structure has been resolved is *Tenebrio molitor* (TMA) α-

(Franco *et al*. 2002; Svensson et al., 2004).

(Hilder et al. 1987).

**2. Insect α-amylases**

as starch or glycogen have been found (Terra et al. 1999).

upon the ionic size ( Terra and Ferreira, 1994).

α-AIs are abundant in microorganisms, higher plants, and animals (Da Silva et al., 2000; Toledo et al., 2007). These organisms produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. α-Amylase inhibitors can be extracted from several plant species including legumes (Marshall and Lauda, 1975; Ishimoto et al., 1996; Grossi-de-Sa et al., 1997) and cereals (Abe et al., 1993; Feng et al., 1996; Yamagata et al., 1998; Franco et al., 2000; Iulek et al., 2000). Diverse α-amylase inhibitors reveal different characteristics against various α-amylases (Franco et al., 2002). In Table 1 inhibitory activity of αAIs from different sources are reviewed. α-AIs are naturally used by plants as a defense mechanism against insect pests (Ishimoto et al., 1989; Kluh et al., 2005). Moreover, there is a great interest to use α-AIs for control of insect pest and also to use them for production of transgenic plants that are resistant against insect pest (Gatehouse et al., 1998; Chrispeels et al., 1998; Gatehouse and Gatehouse, 1998; Valencia et al., 2000; Yamada et al., 2001). Inhibitors to insect α-amylase have already been demonstrated to be an important biological system in the control of insect pests (Franco et al., 2002; Carlini and Grossi-de-Sa´, 2002; Svensson *et al*., 2004). The expression of α-amylase inhibitors has been showed to be effective in transgenic plants. The expression of the cDNA encoding αAl-I into some plants such as pea (*Pisum sativum* L.) and azuki bean (*Vigna anguralis* L.) against bruchid beetle pests (Coleoptera: Bruchidae) has been well documented for showing ability of these inhibitors to be used as plant resistance factors against some species of insect pests (Ishimoto et al., 1989; Yamada et al., 2001; Kluh et al., 2005). Pea and azuki transgenic plants expressing α-amylase inhibitors from common beans (α-AI) were completely resistant to the *Bruchus pisorum* and *Callosobruchus chinensis* weevils (Morton *et al.* 2000). Rye α-amylase inhibitor expressed in transgenic tobacco seeds (*Nicotiana tabacum*) caused 74% mortality in *Anthonomus grandis* first instar larvae when transgenic seed flour mixture used in artificial diet (Dias et al., 2010).

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control 233

*Anthonomus grandis*

*Eurygaster integriceps Tenebrio molitor Rhyzopcrtha dominica Callosobruchus maculates*

*Acanthoscelides obtectus, Zabrotess subfasciatus Anthonomus grandis*

*Sitophilus oryzae Cylas formicarius elegantulus*

*Sitophilus oryzae Cylas formicarius elegantulus*

*Tribolium castaneum*

*Zabrotes subfasciatus*

Table 1. Plant α-amylase inhibitors and their activities against insect pests (literature review

Based on structural similarity, there are six different proteinaceous α-amylase inhibitors with plant origin including lectin-like, knottin-like, CM-proteins, Kunitz-like, c-purothionin-

*Tribolium castaneum*

*Araecerus fasciculatus* In vitro

*Araecerus fasciculatus* In vitro

*Callosobruchus maculatus* In vitro

*Pterodon Callosobruchus maculatus* In vivo (Silva et al., 2007)

*Callosobruchus maculatus* In vivo (Alves, 2009)

Transge nic plant

In vitro In vivo

In vivo In vitro

**condition Plant origin Target pest α-Amylase**

CpAI *Carica papaya Callosobruchus maculatus* In vivo

0.19 AI *Triticum aestivum Acanthoscelides obtectus* In vivo

**inhibitor**

(AI)-1

*Triticum aestivum*

0.53 AI

TAI1,TAI2 C154, C178, C249, C439, C487

α-ZSAI

PpAl

since 2002).

DR1-DR4 *Delonix regia*

α-AIs from *Triticum aestivum*

BIII *Secale cereale*

SPAI1-SPAI4 *Ipomoea batatas*

α-PPAI and *Ficus* sp.

*Colocasia esculenta*

*pubescens*

**3.1 Plant α-amylase inhibitor classes**

like, and thaumatin-like (Richardson, 1990) (Table 2).

 Plant α-amylase inhibitors and their activities against insect pests (literature review since 2002).

#### **3.1 Plant α-amylase inhibitor classes**

232 New Perspectives in Plant Protection

*Anthonomus grandis Acanthoscelides obtectus, Zabrotess subfasciatus and*

*Tecia solanivora*

*coffee berry borer pest Callosobruchus maculatus Callosobruchus chinensis Zabrotes subfasciatus Sitophilus oryzae Acanthoscelides obtectus Cryptolestes ferrugineus Cryptolestes pusillus Oryzaephilus surinamensis Sitophilus granarius Tribolium castaneum T. castaneum*

*Drosophila melanogaster Sarcophaga bullata Aedes aegypti*

*Monomorium pharaonis*

*Acanthoscelides obtectus and* In vitro

*Zabrotes subfasciatus*

VrD1 *Vigna radiata Tenebrio molitor* In silico (Liu et al., 2006) KPSl *Vigna radiata Callosobruchus Maculatus* In vivo (Wisessing, 2010)

*Apis mellifica Venturia canescens Ephestia cautella E. elutella E. kuehniella Manduca sexta Ostrinia nubilalis Blattella germanica Liposcelis decolor Acheta domesticus Eurydema oleracea Graphosoma lineatum*

*Callosobruchus maculatus, Zabrotes subfasciatus, Tenebrio molitor*

*Hypothenemus hampei* In vitro

**condition Plant origin Target pest α-Amylase**

Baru seed *Dipteryx alata Callosobruchus maculatus* In vivo

*Phaseolus Hypothenemus hampei*

**inhibitor**

Ric c 3

extract

α-AI-1 and α-AI2

αAI-Pc1

αBIII *Secale cereale*

Ric c 1 and *Ricinus communis*

*Phaseolus coccineus*

*coccineus*

α-AI1, α-AI2 *Phaseolus vulgaris*

VuD1 *Vigna unguiculata*

**Reference Test**

2010)

2007)

transgenic tobacco In vitro

Feeding assay

Transge nic plant

Transge nic plant In vivo

(Dias et al., 2005,

(Do Nascimento et al., 2011)

(Bonavides et al.,

(Valencia-Jiménez et al., 2008)

(de Azevedo et al.,2006)

(Barbosa et al., 2010; Solleti et al., 2008; Nishizawa etal., 2007; Ignacimuthu,and Prakash, 2006; Kluh et al., 2005)

(Pelegrini et al.,

2008)

Based on structural similarity, there are six different proteinaceous α-amylase inhibitors with plant origin including lectin-like, knottin-like, CM-proteins, Kunitz-like, c-purothioninlike, and thaumatin-like (Richardson, 1990) (Table 2).

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control 235

disulfide connectivity are related to the squash family of proteinase inhibitors, to the cellulose binding domain of cellobiohydrolase, and to omega-conotoxin, i.e. knottins. The three-dimensional model of AAI contains three antiparallel β strands and it is extremely rich

Kunitz-like α-amylase inhibitors commonly found in cereals such as barley, wheat and rice (Micheelsen et al., 2008; Nielsen et al., 2004). Recently, they have also reported from legums, e.g. Cowpea (*Vigna unguiculata* ) (Alves et al., 2009). Kunitz-like α-amylase inhibitors from Cowpea were active against both insect and mammals α-amylase with different intensity (Alves et al., 2009). α-Amylase/subtilisin inhibitors (BASI) are the most studied inhibitors of the Kunitz-like trypsin inhibitor family (Melo et al., 2002), that have bifunctional action i.e. as a plant defense and also as a regulator of endogenous α-amylase action (Micheelsen et al., 2008; Nielsen et al., 2004). The structure of BASI consists of two disulfide bonds and a 12 stranded β-barrel structure which belongs to the β-trefoil fold family. The interaction of Kunitz-like α-amylase inhibitors with the barley α-amylase 2 (AMY2) revealed a new kind of binding mechanisms of proteinaceous α-amylase inhibitors since calcium ions modulate

The members of this family contain inhibitors with 47 – 48 amino acid residues that show strongly inhibition activity against insect α-amylases (Bloch Jr and Richardson, 1991). SIα-1, SIα-2 and SIα-3 are three isoinhibitors isolated from *Sorghum bicolor* and showed inhibitory activity against digestive a-amylases of cockroach and locust, poorly inhibited *A. oryzae* α-amylases and human saliva. These inhibitors did not show inhibitory activity on the αamylases from porcine pancreas, barley and *Bacillus* sp. (Bloch Jr and Richardson, 1991). The three isoforms contain eight cyctein residues forming four disulfide bonds (Nitti et al., 1995).

 Cereal-type is also refers to these inhibitors since they are present in cereals. CM-proteins show a typical double-headed α-amylase/trypsin domain (Campos and Richardson, 1983). This feature make it possible that they show inhibitory activity against α-amylases (Barber et al., 1986a) and trypsin-like enzymes (Barber et al., 1986b; De Leo et al., 2002) separately or show α-amylases/ trypsin-like inhibitory activity at the same time (Garcia- Maroto et al., 1991). The CM protein family includes lipid transfer proteins (Lerche and Poulsen , 1998; Svensson et al ., 1986) and proteins related to cold tolerance (Hincha, 2002). The α-amylase inhibitor 0.19, one of the most studied inhibitor of this family, has a broad specificity and inhibits α-amylases from insects, birds and mammal (Titarenko et al., 2000; Franco et al., 2000; Franco et al., 2002; Oneda et al., 2004). It has 124 amino-acid residues and acting as a homodimer (Oda et al., 1997; Franco et al., 2000). The X-ray crystallographic analysis of 0.19 AI demonstrated that each subunit is composed of four major α-helices, one one-turn helix, and two short antiparalell β-strands. The subunits in a

in disulfides (Carugo et al., 2001).

the interaction (Melo et al., 2002).

**3.1.4 γ - Purothionin type**

**3.1.5 CM- proteins**

**3.1.3 Kunitz-type**

Table 2. Classification of plant α-amylase inhibitors based on structural similarity (Richardson, 1990).

#### **3.1.1 Lectin-like inhibitors**

There have been particular attentions on lectin-like inhibitors and they are toxic against several insect pests (Ishimoto and Kitamura, 1989; Huesing et al., 1991a; Ishimoto and Chrispeels, 1996; Grossi-de-Sa et al., 1997, Kluh et al,., 2005; Karbache et al., 2011). αAI-1 and αAI-2, Two lectin-like inhibitors, were identied in common white, red and black kidney beans (Ishimoto and Chrispeels, 1996;). They show different specificity against α-amylases because of the mutation in their primary structure (Grossi de Sa et al., 1997). αAI-1 inhibits mammalian α-amylases and several insect amylases, but it is not active against Mexican bean weevil (*Zabrotes subfasciatus*). On the other hand, αAI-2 does not inhibit the α-amylases recognized by αAI-1 but inhibits the α–amylase of *Z. subfasciatus* (Ishimoto an d Chrispeels, 1996; Kluh et al., 2005).

#### **3.1.2 Knottin-like inhibitors**

The major α-amylase inhibitor (AAI) present in the seeds of *Amaranthus hypocondriacus*, is a 32-residue-long polypeptide with three disulfide bridges (Chagolla-Lopez et al., 1996). AAI strongly inhibits α-amylase activity of *Tribolium castaneum* and *Prostephanus truncates*, however, does not inhibit proteases and mammalian α-amylases. AAI is the smallest prot einaceous inhibitor of a-amylases yet described. Its residue conservation patterns and disulfide connectivity are related to the squash family of proteinase inhibitors, to the cellulose binding domain of cellobiohydrolase, and to omega-conotoxin, i.e. knottins. The three-dimensional model of AAI contains three antiparallel β strands and it is extremely rich in disulfides (Carugo et al., 2001).

#### **3.1.3 Kunitz-type**

234 New Perspectives in Plant Protection

**number (aa)**

Insect, 240-250 αAI1, αAI2

**Plant Target**

Mammalian, Fungal

Knottin- Amaranth Insect 32 AAI

Wheat, Insect, Plant 176-181

Insect, 47-48

Insect, 124-160

Table 2. Classification of plant α-amylase inhibitors based on structural similarity

There have been particular attentions on lectin-like inhibitors and they are toxic against several insect pests (Ishimoto and Kitamura, 1989; Huesing et al., 1991a; Ishimoto and Chrispeels, 1996; Grossi-de-Sa et al., 1997, Kluh et al,., 2005; Karbache et al., 2011). αAI-1 and αAI-2, Two lectin-like inhibitors, were identied in common white, red and black kidney beans (Ishimoto and Chrispeels, 1996;). They show different specificity against α-amylases because of the mutation in their primary structure (Grossi de Sa et al., 1997). αAI-1 inhibits mammalian α-amylases and several insect amylases, but it is not active against Mexican bean weevil (*Zabrotes subfasciatus*). On the other hand, αAI-2 does not inhibit the α-amylases recognized by αAI-1 but inhibits the α–amylase of *Z. subfasciatus* (Ishimoto an d Chrispeels,

The major α-amylase inhibitor (AAI) present in the seeds of *Amaranthus hypocondriacus*, is a 32-residue-long polypeptide with three disulfide bridges (Chagolla-Lopez et al., 1996). AAI strongly inhibits α-amylase activity of *Tribolium castaneum* and *Prostephanus truncates*, however, does not inhibit proteases and mammalian α-amylases. AAI is the smallest prot einaceous inhibitor of a-amylases yet described.  Recently, they have also reported from legums, e.g. Cowpea (*Vigna unguiculata* ) (Alves et al., 2009). Kunitz-like α-amylase inhibitors from Cowpea were active against both insect and mammals α-amylase with different intensity (Alves et al., 2009). α-Amylase/subtilisin inhibitors (BASI) are the most studied inhibitors of the Kunitz-like trypsin inhibitor family (Melo et al., 2002), that have bifunctional action i.e. as a plant defense and also as a regulator of endogenous α-amylase action (Micheelsen et al., 2008; Nielsen et al., 2004). The structure of BASI consists of two disulfide bonds and a 12 stranded β-barrel structure which belongs to the β-trefoil fold family. The interaction of Kunitz-like α-amylase inhibitors with the barley α-amylase 2 (AMY2) revealed a new kind of binding mechanisms of proteinaceous α-amylase inhibitors since calcium ions modulate the interaction (Melo et al., 2002).

#### **3.1.4 γ - Purothionin type**

The members of this family contain inhibitors with 47 – 48 amino acid residues that show strongly inhibition activity against insect α-amylases (Bloch Jr and Richardson, 1991). SIα-1, SIα-2 and SIα-3 are three isoinhibitors isolated from *Sorghum bicolor* and showed inhibitory activity against digestive a-amylases of cockroach and locust, poorly inhibited *A. oryzae* α-amylases and human saliva. These inhibitors did not show inhibitory activity on the αamylases from porcine pancreas, barley and *Bacillus* sp. (Bloch Jr and Richardson, 1991).  Cereal-type is also refers to these inhibitors since they are present in cereals. CM-proteins show a typical double-headed α-amylase/trypsin domain (Campos and Richardson, 1983). This feature make it possible that they show inhibitory activity against α-amylases (Barber et al., 1986a) and trypsin-like enzymes (Barber et al., 1986b; De Leo et al., 2002) separately or show α-amylases/ trypsin-like inhibitory activity at the same time (Garcia- Maroto et al., 1991). The CM protein family includes lipid transfer proteins (Lerche and Poulsen , 1998; Svensson et al ., 1986) and proteins related to cold tolerance (Hincha, 2002). The α-amylase inhibitor 0.19, one of the most studied inhibitor of this family, has a broad specificity and inhibits α-amylases from insects, birds and mammal (Titarenko et al., 2000; Franco et al., 2000; Franco et al., 2002; Oneda et al., 2004). It has 124 amino-acid residues and acting as a homodimer (Oda et al., 1997; Franco et al., 2000). The X-ray crystallographic analysis of 0.19 AI demonstrated that each subunit is composed of four major α-helices, one one-turn helix, and two short antiparalell β-strands. The subunits in a

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control 237

with other functions in insect (Matsumoto et al., 1997). Cysteine proteinases have their optimum activity in the alkaline range (Bode and Huber, 1992; Oliveira et al., 2003). It has been revealed that cathepsin L-like enzymes are the only quantitatively important member of cysteine proteinases presented in midgut of insects. Digestive cathepsin L-like enzymes have been purified from *Diabrotica virgifera* (Coleoptera: Cucujiformia) (Koiwa et al., 2000), *Acyrthosiphon pisum* (Hemiptera: Sternorrhyncha) (Cristofoletti et al., 2003), *T. molitor* (Coleoptera: Cucujiformia) (Cristofoletti et al. , 2005), *Sphenophorus levis (*Coleoptera: Curculionidae) (Soares-Costa et al., 2011; Fonseca et al., 2012), and *Triatoma brasiliensis*

PIs are a natural plant defensive mechanism against insect herbivores which were viewed as promising compounds for developing insect resistance transgenic crops that over-express PIs (Gatehouse, 2011). PIs have found in animals, plants (particularly legumes and cereals), and microorganisms. Most storage organs such as seeds and tubers contain 1-10% of their total proteins as PIs with different biochemical and structural properties inhibiting different types of proteases (Volpicella et al., 2011). PIs play an important role in different physiological functions of plants including as storage proteins, and regulators of endogenous proteolytic activity (Ryan, 1990), modulators of apoptotic processes or programmed cell death (Solomon et al., 1999), and defense components associated with the resistance of plants against insects and pathogens (Lu et al., 1998; Pernas et al., 1999). Green and Ryan (1972) pioneer works revealed the roles of PIs in the plant-insect interaction. They showed induction of plant PIs in response to attack of insects and pathogen and named this induction as "defense-response" of the plant against the pests. Production of PIs that inhibit digestive herbivore gut proteases inspired the eld of plant– insect interactions and became an outlandish example of induced plant defenses. Since then, several PIs of insect proteinases have been identified and characterized (Garcia-Olmedo et al. 1987; Lawrence and Koundal 2002). Despite insects that feed on sap or seeds, most phytophagous insects are nutritionally limited by protein digestion.Since plant tissues are nitrogen deficient compared to insect composition, and the main source of nitrogen available to the insect is protein (Gatehouse, 2011). Therefore, their proteinases have an important role in digestion of proteins and maintaining of needed nitrogen. Inactivation of digestive enzymes by PIs results in blocking of gut proteinases that leads to poor nutrient utilization, retarded development, and death because of starvation

There have been considerable number of reviews on plant PIs describing their classification (Turra et al., 2011; Volpicella et al., 2011), biochemical and structural properties (Antao and Malcata, 2005; Bateman and James, 2011; Oliva et al., 2011), their role in plant physiology (Schaller, 2004; Salas et al., 2008; Roberts and Hejgaard, 2008), insect-plant co-evolution (Jongsma and Beekwilder, 2011), and their application in different areas including pest control (Lawrence and Koundal, 2002; Gatehouse, 2011), nutritional (Clemente et al., 2011)

PIs are classied based on the type of enzyme they inhibit: Serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, or metallocarboxy-protease

(Reduviidae, Triatominae) (Waniek et al., 2012).

(Jongsma and Bolter 1997; Gatehouse and Gatehouse, 1999).

as well as pharmaceutical (Gomes et al., 2011) applications.

**5.1 Plant proteinase inhibitors classes**

**5. Proteinase inhibitors from plants**

dimer are related each other by non-crystallographic 2-fold axis, and the interface is mainly composed of hydrophobic residues (Oda et al., 1997).

#### **3.1.6 Thaumatin-like**

This family contains proteins with molecular weight about 22 kDa, which are homologous with the intensely sweet protein thaumatin from fruits of *Thaumatococcw daniellii* Benth, thus they are called thaumatin-like (Cornelissen et al., 1986; Vigers., 1991; Hejgaard et al., 1991). Although thaumatin-like proteins is a homologue of the sweet protein thaumatin and exhibit α-amylase inhibitory activity, however, thaumatin and other related proteins do not show inhibitory activity against α-amylases (Franco et al., 2002; Svensson et al., 2004 and references therein). Zeamatin from maize is the best-characterized member of this family which inhibits insect but not mammalian α-amylases. Zeamatin has 13 β strands, 11 of which form a β sandwich at the core of protein (Batalia et al., 1996). Zeamatin has been applied as antifungal drugs because it binds to β-1,3-glucan and permeabilizes fungal cells resulting in cell death (Roberts and Selitrennikoff, 1990; Franco et al., 2000).

#### **4. Insect digestive proteinases**

Proteinases, which are also known as endopeptidases, enroll an important function in protein digestion. These enzymes begin the protein digestion process by breaking internal bonds in proteins. The amino acid residues vary along the peptide chain, therefore, different kind of proteinases are necessary to hydrolyze them. Based on active site group and their correspond mechanism, digestive proteinases can be classified as serine, cysteine, and aspartic proteinases (Terra and Ferreira, 2012). Serine, cysteine are the most widespread proteinases in insect digestive system.

Serine proteinases (EC 3.4.21) have the active site composed of serine, histidine, and aspartic acid residues (also called catalytic triad). Trypsin (EC <IP_ADDRESS>), chymotrypsin (EC <IP_ADDRESS>), and elastase (EC <IP_ADDRESS>) are the major digestive enzymes of this family that usually work at alkhalin pH. These enzymes differ in structural features that are associated with their different substrate specificities. Trypsin are endoproteases that attack proteins at residues of arginine and lysine. Generally, insect trypsins have molecular masses in the range 20–35 kDa, pI values 4–5, and pH optima 8–10 (Terra and Ferreira, 2012). Trypsin occurs in the majority of insects, with the remarkable exception of some hemipteran species and some taxa belonging to the series Cucujiformia of Coleoptera like Curculionidae (Terra and Ferreira, 1994). Nevertheless, some heteropteran Hemiptera have trypsin in the salivary glands (Zeng et al. , 2002). Chymotrypsin enzymes attack proteins at aromatic residues (e.g., tryptophan). Insect chymotrypsins usually have molecular masses of 20–30 kDa and pH optima of 8–11 (Terra and Ferreira, 1994). Similar to trypsin, chymotrypsin is also distributed in the majority of insects (Terra and Ferreira, 1994), including those purified from Lepidoptera (Peterson et al., 1995; Volpicella et al., 2006), Diptera (de Almeida et al., 2003; Ramalho-Or tigão et al., 2003), Hemiptera (Colebatch et al., 2002), Hymenoptera (Whitwor th et al., 1998), Siphonaptera (Gaines et al., 1999) and Coleoptera (Oliveira-Neto et al., 2004; Elpidina et al., 2005).

Cysteine proteinases occur in the digestive system of insects (Rawlings and Barrett, 1993). These enzymes are also found in other tissue of insects, indicating that they are associated with other functions in insect (Matsumoto et al., 1997). Cysteine proteinases have their optimum activity in the alkaline range (Bode and Huber, 1992; Oliveira et al., 2003). It has been revealed that cathepsin L-like enzymes are the only quantitatively important member of cysteine proteinases presented in midgut of insects. Digestive cathepsin L-like enzymes have been purified from *Diabrotica virgifera* (Coleoptera: Cucujiformia) (Koiwa et al., 2000), *Acyrthosiphon pisum* (Hemiptera: Sternorrhyncha) (Cristofoletti et al., 2003), *T. molitor* (Coleoptera: Cucujiformia) (Cristofoletti et al. , 2005), *Sphenophorus levis (*Coleoptera: Curculionidae) (Soares-Costa et al., 2011; Fonseca et al., 2012), and *Triatoma brasiliensis* (Reduviidae, Triatominae) (Waniek et al., 2012).

#### **5. Proteinase inhibitors from plants**

236 New Perspectives in Plant Protection

dimer are related each other by non-crystallographic 2-fold axis, and the interface is mainly

This family contains proteins with molecular weight about 22 kDa, which are homologous with the intensely sweet protein thaumatin from fruits of *Thaumatococcw daniellii* Benth, thus they are called thaumatin-like (Cornelissen et al., 1986; Vigers., 1991; Hejgaard et al., 1991). Although thaumatin-like proteins is a homologue of the sweet protein thaumatin and exhibit α-amylase inhibitory activity, however, thaumatin and other related proteins do not show inhibitory activity against α-amylases (Franco et al., 2002; Svensson et al., 2004 and references therein). Zeamatin from maize is the best-characterized member of this family which inhibits insect but not mammalian α-amylases. Zeamatin has 13 β strands, 11 of which form a β sandwich at the core of protein (Batalia et al., 1996). Zeamatin has been applied as antifungal drugs because it binds to β-1,3-glucan and permeabilizes fungal cells

Proteinases, which are also known as endopeptidases, enroll an important function in protein digestion. These enzymes begin the protein digestion process by breaking internal bonds in proteins. The amino acid residues vary along the peptide chain, therefore, different kind of proteinases are necessary to hydrolyze them. Based on active site group and their correspond mechanism, digestive proteinases can be classified as serine, cysteine, and aspartic proteinases (Terra and Ferreira, 2012). Serine, cysteine are the most widespread

Serine proteinases (EC 3.4.21) have the active site composed of serine, histidine, and aspartic acid residues (also called catalytic triad). Trypsin (EC <IP_ADDRESS>), chymotrypsin (EC <IP_ADDRESS>), and elastase (EC <IP_ADDRESS>) are the major digestive enzymes of this family that usually work at alkhalin pH. These enzymes differ in structural features that are associated with their different substrate specificities. Trypsin are endoproteases that attack proteins at residues of arginine and lysine. Generally, insect trypsins have molecular masses in the range 20–35 kDa, pI values 4–5, and pH optima 8–10 (Terra and Ferreira, 2012). Trypsin occurs in the majority of insects, with the remarkable exception of some hemipteran species and some taxa belonging to the series Cucujiformia of Coleoptera like Curculionidae (Terra and Ferreira, 1994). Nevertheless, some heteropteran Hemiptera have trypsin in the salivary glands (Zeng et al. , 2002). Chymotrypsin enzymes attack proteins at aromatic residues (e.g., tryptophan). Insect chymotrypsins usually have molecular masses of 20–30 kDa and pH optima of 8–11 (Terra and Ferreira, 1994). Similar to trypsin, chymotrypsin is also distributed in the majority of insects (Terra and Ferreira, 1994), including those purified from Lepidoptera (Peterson et al., 1995; Volpicella et al., 2006), Diptera (de Almeida et al., 2003; Ramalho-Or tigão et al., 2003), Hemiptera (Colebatch et al., 2002), Hymenoptera (Whitwor th et al., 1998), Siphonaptera (Gaines et al., 1999) and Coleoptera (Oliveira-

Cysteine proteinases occur in the digestive system of insects (Rawlings and Barrett, 1993). These enzymes are also found in other tissue of insects, indicating that they are associated

resulting in cell death (Roberts and Selitrennikoff, 1990; Franco et al., 2000).

composed of hydrophobic residues (Oda et al., 1997).

**3.1.6 Thaumatin-like**

**4. Insect digestive proteinases**

proteinases in insect digestive system.

Neto et al., 2004; Elpidina et al., 2005).

PIs are a natural plant defensive mechanism against insect herbivores which were viewed as promising compounds for developing insect resistance transgenic crops that over-express PIs (Gatehouse, 2011). PIs have found in animals, plants (particularly legumes and cereals), and microorganisms. Most storage organs such as seeds and tubers contain 1-10% of their total proteins as PIs with different biochemical and structural properties inhibiting different types of proteases (Volpicella et al., 2011). PIs play an important role in different physiological functions of plants including as storage proteins, and regulators of endogenous proteolytic activity (Ryan, 1990), modulators of apoptotic processes or programmed cell death (Solomon et al., 1999), and defense components associated with the resistance of plants against insects and pathogens (Lu et al., 1998; Pernas et al., 1999). Green and Ryan (1972) pioneer works revealed the roles of PIs in the plant-insect interaction. They showed induction of plant PIs in response to attack of insects and pathogen and named this induction as "defense-response" of the plant against the pests. Production of PIs that inhibit digestive herbivore gut proteases inspired the eld of plant– insect interactions and became an outlandish example of induced plant defenses. Since then, several PIs of insect proteinases have been identified and characterized (Garcia-Olmedo et al. 1987; Lawrence and Koundal 2002). Despite insects that feed on sap or seeds, most phytophagous insects are nutritionally limited by protein digestion.Since plant tissues are nitrogen deficient compared to insect composition, and the main source of nitrogen available to the insect is protein (Gatehouse, 2011). Therefore, their proteinases have an important role in digestion of proteins and maintaining of needed nitrogen. Inactivation of digestive enzymes by PIs results in blocking of gut proteinases that leads to poor nutrient utilization, retarded development, and death because of starvation (Jongsma and Bolter 1997; Gatehouse and Gatehouse, 1999).

There have been considerable number of reviews on plant PIs describing their classification (Turra et al., 2011; Volpicella et al., 2011), biochemical and structural properties (Antao and Malcata, 2005; Bateman and James, 2011; Oliva et al., 2011), their role in plant physiology (Schaller, 2004; Salas et al., 2008; Roberts and Hejgaard, 2008), insect-plant co-evolution (Jongsma and Beekwilder, 2011), and their application in different areas including pest control (Lawrence and Koundal, 2002; Gatehouse, 2011), nutritional (Clemente et al., 2011) as well as pharmaceutical (Gomes et al., 2011) applications.

#### **5.1 Plant proteinase inhibitors classes**

PIs are classied based on the type of enzyme they inhibit: Serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, or metallocarboxy-protease

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control 239

levels of chymotrypsin resistant to inhibition by NaPI. In order to solve this problem, NaPI was expressed in synergism with *Solanum tuberosum* potato type I inhibitor (StPin1A), which strongly inhibited NaPI-resistant chymotrypsins. The mutual inhibitory effect of NaPI and StPin1A on *H. armigera* larval growth was observed both in laboratory conditions as well as

Iimproved crop protection achieved using mixtures of inhibitors in which one class of proteinase inhibitor is utilized to contest the genetic ability of an insect to adapt to a additional class of proteinase inhibitor. Furthermore, amylase inhibitors have also been utilized as defense factors against insects in genetic modified plants. Several amylase inhibitors have been expressed in different plants. However the expression of -amylase inhibitors (α-AI) from scarlet runner bean (*Phaseolus coccineus*) and common bean (*Phaseolus vulgaris*) has been extremely protective in genetic modified plants, showing enhanced shelter against pea weevils (Shade et al., 1994; Schroeder et al., 1995), adzuki bean (Ishimoto et al., 1996), chickpea (Sarmah et al., 2004; Ignacimuthu et al., 2006, Campbell et al., 2011) and cowpea (Solleti et al., 2008). Furthermore, transgenic pea showed enhanced defense against the pea weevil *Bruchus pisorum* was shown under field conditions (Morton et al., 2000). All these trials associated the α-AI expression with the seed-specific promoter of

Moreover other crops, in addition to legumes, were also transformed with amylase inhibitors. The Rubiacea *Coffea arabica* was also engineered with -AI1 under control of phytohemagglutinin promoter (Barbosa et al., 2010). The presence of this gene was observed by PCR and Southern blotting in six regenerated transgenic T1 coffee plants. Iimmunoblotting and ELISA experiments using antibodies against -AI1 revealed the presence of this inhibitor at a concentration of 0.29 % in seed extracts. The presence of this inhibitor was able to cause a clear inhibitory activity on digestive enzymes of *Hypotenemus*

Also, an -amylase inhibitor from cereal-family (BIII) from rye (*Secale cereale*) seeds was also cloned and expressed initially in *E. coli* showing clear activity toward -amylases of larvae of the coleopteran pests *Acanthoscelides obtectus*, *Zabrotess subfasciatus* and *Anthonomus grandis* (Dias et al. 2005). BIII inhibitor was also expressed under control of phytohemaglutinin promoter in tobacco plants (*Nicotiana tabacum*). Besides, the occurrence of BIII-rye gene and further protein expression were confirmed. Immunological analyzes indicated that the recombinant inhibitor was produced in concentration ranging from 0.1% to 0.28% (w: w). Bioassays using transgenic seed flour for artificial diet caused 74% mortality for cotton boll weevil *A. grandis* suggesting that rye inhibitor could be an auspicious biotechnological tool for yield transgenic cotton plants with an improved resistance to

While important protection against insect pests has been routinely achieved, the transgenic plants do not show levels of resistance considered commercially possible. As a consequence of selective pressures, insect herbivores have developed various adaptation mechanisms to overcome the defensive effects of plant inhibitors. Common polyphagous crop pests are well adapted to avoid a wide range of different inhibitors, which have only limited effects.

in field trials of transgenic plants.

phytohemagglutinin from *P. vulgaris*.

*hampei* suggesting a possible protective effect.

weevil (Dias et al., 2010).

**7. Summary**

inhibitors (Ryan, 1990; Mosolov, 1998; Bode and Huber, 2000). Plant serine proteinase inhibitors further sub-classified to a number of subfamilies based on their amino acid sequences and structural properties known as Kunitz type, Bowman-Birk type, Potato I type, and Potato II type inhibitors (Bode and Huber, 1992). The families of PIs could not, however, be grouped on the basis of the catalytic type of enzymes inhibited, since a number of families contain cross-class inhibitors. Despite cysteine and metallocarboxy inhibitor families, all other reported families of PIs contain inhibitors of serin proteases. (Volpicella et al., 2002). The proteins in Kunitz-like family, for instance, generally inhibit serine proteinases, besides they also include inhibitors of cysteine and aspartate proteases (Heibges et al., 2003). There are some exceptions, however, that PIs families may have not inhibitors of serine proteases such as aspartic protease inhibitors in Kunitz and cystein families and also potato cystein protease inhibitors that belongs to Kunitz family (Volpicella et al., 2002).

#### **6. Transgenic plants expressing digestive enzyme inhibitors**

It seems obvious that the prospective amylase and proteinase inhibitors can function as a biotechnological tool for the discovery of novel bioinsecticides or in the construction of transgenic plants with enhanced resistance toward pests and pathogens.

Since Johnson et al. (1989) expressed proteinase inhibitors in transgenic tobacco providing enhanced resistance against *Manduca sexta* larvae, hundreds of reports have been produced in this specific issue. As previously described, proteinase inhibitors could act on the digestive enzymes of insect herbivores reducing food digestibility. Attempts to achieve this defense mechanism in plants, genetic engineering have used over-expression of both exogenous and endogenous proteinase inhibitors (Gatehouse, 2011).

Among several targets, Lepidopteran has been clearly focused, since they are important groups of crop insect-pests in the world. Until now the only commercially accessible transgenes for control of these insect pests encode Cry *Bacillus thuringiensis* (Bt) toxins and the Vip3Aa20 toxin (United States Environmental Protection Agency, 2009). Several trials have been performed by using proteinase inhibitors. For example the mustard trypsin inhibitor (MTI-2) was expressed at different levels in transgenic tobacco, Arabidopsis and oilseed rape lines. The three plants were challenged against different lepidopteran insectpests, including *Plutella xylostella* (L.), which was extremely sensible to MTI-2 ingestion being completely exterminated (de Leo et al., 2001). Furthermore MTI-2 was also expressed at different levels in transgenic tobacco lines and was further appraised by feeding of the lepidopteran larvae, *Spodoptera littoralis* (de Leo and Galerani et al., 2002). A surprising result was obtained. *S. littoralis* larvae feed on transgenic tobacco expressing MTI-2 were unaffected.However, significant reduction on fertility was obtained suggesting that multiple effects could be obtained with a single proteinase inhibitor. In this view, several research groups have produced and evaluated transgenic plants synthesizing proteinase inhibitors and attacked by Lepidoptera pests. Among inhibitors expressed in transgenic plants were NaPI, the *Nicotiana alata* proteinase inhibitor and also the multidomain potato type II inhibitor that is produced at enhanced levels in the female reproductive organs of *N. alata* (Dunse et al., 2010). The individual inhibitory domains of NaPI target trypsin and chymotrypsin, from digestive tract of lepidopteran larval pests. While feeding on NaPI, dramatically reduced the *Helicoverpa punctigera* growth, surviving larvae exhibited high levels of chymotrypsin resistant to inhibition by NaPI. In order to solve this problem, NaPI was expressed in synergism with *Solanum tuberosum* potato type I inhibitor (StPin1A), which strongly inhibited NaPI-resistant chymotrypsins. The mutual inhibitory effect of NaPI and StPin1A on *H. armigera* larval growth was observed both in laboratory conditions as well as in field trials of transgenic plants.

Iimproved crop protection achieved using mixtures of inhibitors in which one class of proteinase inhibitor is utilized to contest the genetic ability of an insect to adapt to a additional class of proteinase inhibitor. Furthermore, amylase inhibitors have also been utilized as defense factors against insects in genetic modified plants. Several amylase inhibitors have been expressed in different plants. However the expression of -amylase inhibitors (α-AI) from scarlet runner bean (*Phaseolus coccineus*) and common bean (*Phaseolus vulgaris*) has been extremely protective in genetic modified plants, showing enhanced shelter against pea weevils (Shade et al., 1994; Schroeder et al., 1995), adzuki bean (Ishimoto et al., 1996), chickpea (Sarmah et al., 2004; Ignacimuthu et al., 2006, Campbell et al., 2011) and cowpea (Solleti et al., 2008). Furthermore, transgenic pea showed enhanced defense against the pea weevil *Bruchus pisorum* was shown under field conditions (Morton et al., 2000). All these trials associated the α-AI expression with the seed-specific promoter of phytohemagglutinin from *P. vulgaris*.

Moreover other crops, in addition to legumes, were also transformed with amylase inhibitors. The Rubiacea *Coffea arabica* was also engineered with -AI1 under control of phytohemagglutinin promoter (Barbosa et al., 2010). The presence of this gene was observed by PCR and Southern blotting in six regenerated transgenic T1 coffee plants. Iimmunoblotting and ELISA experiments using antibodies against -AI1 revealed the presence of this inhibitor at a concentration of 0.29 % in seed extracts. The presence of this inhibitor was able to cause a clear inhibitory activity on digestive enzymes of *Hypotenemus hampei* suggesting a possible protective effect.

Also, an -amylase inhibitor from cereal-family (BIII) from rye (*Secale cereale*) seeds was also cloned and expressed initially in *E. coli* showing clear activity toward -amylases of larvae of the coleopteran pests *Acanthoscelides obtectus*, *Zabrotess subfasciatus* and *Anthonomus grandis* (Dias et al. 2005). BIII inhibitor was also expressed under control of phytohemaglutinin promoter in tobacco plants (*Nicotiana tabacum*). Besides, the occurrence of BIII-rye gene and further protein expression were confirmed. Immunological analyzes indicated that the recombinant inhibitor was produced in concentration ranging from 0.1% to 0.28% (w: w). Bioassays using transgenic seed flour for artificial diet caused 74% mortality for cotton boll weevil *A. grandis* suggesting that rye inhibitor could be an auspicious biotechnological tool for yield transgenic cotton plants with an improved resistance to weevil (Dias et al., 2010).

#### **7. Summary**

238 New Perspectives in Plant Protection

inhibitors (Ryan, 1990; Mosolov, 1998; Bode and Huber, 2000). Plant serine proteinase inhibitors further sub-classified to a number of subfamilies based on their amino acid sequences and structural properties known as Kunitz type, Bowman-Birk type, Potato I type, and Potato II type inhibitors (Bode and Huber, 1992). The families of PIs could not, however, be grouped on the basis of the catalytic type of enzymes inhibited, since a number of families contain cross-class inhibitors. Despite cysteine and metallocarboxy inhibitor families, all other reported families of PIs contain inhibitors of serin proteases. (Volpicella et al., 2002). The proteins in Kunitz-like family, for instance, generally inhibit serine proteinases, besides they also include inhibitors of cysteine and aspartate proteases (Heibges et al., 2003). There are some exceptions, however, that PIs families may have not inhibitors of serine proteases such as aspartic protease inhibitors in Kunitz and cystein families and also potato cystein protease inhibitors that belongs to Kunitz family (Volpicella

It seems obvious that the prospective amylase and proteinase inhibitors can function as a biotechnological tool for the discovery of novel bioinsecticides or in the construction of

Since Johnson et al. (1989) expressed proteinase inhibitors in transgenic tobacco providing enhanced resistance against *Manduca sexta* larvae, hundreds of reports have been produced in this specific issue. As previously described, proteinase inhibitors could act on the digestive enzymes of insect herbivores reducing food digestibility. Attempts to achieve this defense mechanism in plants, genetic engineering have used over-expression of both

Among several targets, Lepidopteran has been clearly focused, since they are important groups of crop insect-pests in the world. Until now the only commercially accessible transgenes for control of these insect pests encode Cry *Bacillus thuringiensis* (Bt) toxins and the Vip3Aa20 toxin (United States Environmental Protection Agency, 2009). Several trials have been performed by using proteinase inhibitors. For example the mustard trypsin inhibitor (MTI-2) was expressed at different levels in transgenic tobacco, Arabidopsis and oilseed rape lines. The three plants were challenged against different lepidopteran insectpests, including *Plutella xylostella* (L.), which was extremely sensible to MTI-2 ingestion being completely exterminated (de Leo et al., 2001). Furthermore MTI-2 was also expressed at different levels in transgenic tobacco lines and was further appraised by feeding of the lepidopteran larvae, *Spodoptera littoralis* (de Leo and Galerani et al., 2002). A surprising result was obtained. *S. littoralis* larvae feed on transgenic tobacco expressing MTI-2 were unaffected.However, significant reduction on fertility was obtained suggesting that multiple effects could be obtained with a single proteinase inhibitor. In this view, several research groups have produced and evaluated transgenic plants synthesizing proteinase inhibitors and attacked by Lepidoptera pests. Among inhibitors expressed in transgenic plants were NaPI, the *Nicotiana alata* proteinase inhibitor and also the multidomain potato type II inhibitor that is produced at enhanced levels in the female reproductive organs of *N. alata* (Dunse et al., 2010). The individual inhibitory domains of NaPI target trypsin and chymotrypsin, from digestive tract of lepidopteran larval pests. While feeding on NaPI, dramatically reduced the *Helicoverpa punctigera* growth, surviving larvae exhibited high

**6. Transgenic plants expressing digestive enzyme inhibitors**

transgenic plants with enhanced resistance toward pests and pathogens.

exogenous and endogenous proteinase inhibitors (Gatehouse, 2011).

et al., 2002).