Diverse forms of Pin-II family proteinase inhibitors from Capsicum annuum adversely affect the growth and development of Helicoverpa armigera

Novel forms of Pin-II type proteinase inhibitor (PIs) cDNAs (CanPIs) having three or four inhibitory repeat domains (IRD) were isolated from the developing green fruits of Capsicum annuum. Deduced amino acid (aa) sequences of the CanPIs showed up to 15% sequence divergence among each other or reported inhibitors (CanPI-1AF039398, CanPI-2AF221097). Amino acid sequence analysis of these CanPIs revealed that three IRD PIs have trypsin inhibitory sites, while four IRD CanPIs have both trypsin and chymotrypsin inhibitory sites. Four CanPIs, two having three IRD (CanPI-3AY986465 and CanPI-5DQ005912) and two having four IRD (CanPI-7DQ005913 and CanPI-9DQ005915), were cloned in Pichia pastoris to express recombinant CanPIs. Recombinant CanPIs inhibited 90% of bovine trypsin (TI), while chymotrypsin inhibition (CI) varied with the number of chymotrypsin inhibitory sites in the CanPIs. Recombinant inhibitors inhibited over 70% of the gut proteinase activity of Helicoverpa armigera. H. armigera larvae fed on recombinant CanPIs individually incorporated into artificial diet, showed 35% mortality; in addition, weight gain in H. armigera larvae and pupae was severely reduced compared to controls. Of the four CanPIs, CanPI-7, which has two sites for TI and CI, was the only one to have a consistently antagonistic effect on H. armigera growth and development. We conclude that among the four recombinant PIs tested, CanPIs containing diverse IRDs are best suited for developing insect-resistant transgenic plants.     

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.     

Structural refinement of insecticidal plant proteinase inhibitors from Nicotiana alata

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.     

Effects of class-specific,synthetic, and natural proteinase inhibitors on life-history traits of the cotton bollworm Helicoverpa armigera

Herbivorous insects have more difficulty obtaining proteins from their food than do predators and parasites. The scarcity of proteins in their diet requires herbivores to feed voraciously, thus heavily damaging their host plants. Plants respond to herbivory by producing defense compounds, which reduce insect growth, retard development, and increase mortality. Herbivores use both pre- and postdigestive response mechanisms to detect and avoid plant defense compounds. Proteinase inhibitors (PIs) are one example of plant compounds produced as a direct defense against herbivory. Many insects can adapt to PIs when these are incorporated into artificial diets. However, little is known about the effect of PIs on diet choice and feeding behavior. We monitored the diet choice, life-history traits, and gut proteinase activity of Helicoverpa armigera larvae using diets supplemented with synthetic and natural PIs. In choice experiments, both neonates and fourth-instar larvae preferred the control diet over PI-supplemented diets, to varying degrees. Larvae that fed on PI-supplemented diets weighed less than those that fed on the control diet and produced smaller pupae. Trypsin-specific PIs had a stronger effect on mean larval weight than did other PIs. A reduction of trypsin activity but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets. Therefore, behavioral avoidance of feeding on plant parts high in PIs could be an adaptation to minimize the impact of this plant's defensive strategy.     

Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family.

Six small molecular mass, wound-inducible trypsin and chymotrypsin inhibitor proteins from tobacco (Nicotiana tabacum) leaves were isolated to homogeneity. The isoinhibitors, cumulatively called tobacco trypsin inhibitor (TTI), have molecular masses of approximately 5500 to 5800 D, calculated from gel filtration analysis and amino acid content. The amino acid sequence of the entire 53 residues of one isoinhibitor, TTI-1, and the sequence of 36 amino acid residues from the N terminus of a second isoinhibitor, TTI-5, were determined. The two isoinhibitors differ only at residue 11, which is threonine in TTI-1 and lysine in TTI-5. The isoinhibitors are members of the potato inhibitor II family and show considerable identity with the small molecular mass members of this family, which include the eggplant inhibitor, two small molecular mass trypsin and chymotrypsin inhibitors from potatoes, and an inhibitor from pistils of the ornamental plant Nicotiana alata. Antibodies produced against the isoinhibitors in rabbits were used in radial immunoassays to quantify both the systemic wound inducibility of TTI in tobacco leaves and its constitutive levels in flowers.     

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases

Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.     

Compensatory proteolytic responses to dietary proteinase inhibitors from Albizia lebbeck seeds in the Helicoverpa armigera larvae

Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies.     

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.     

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut

Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.     

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests.     

Abscisic acid and jasmonic acid affect proteinase inhibitor activities in barley leaves

Proteinase inhibitor (PI) accumulation has been described as a plant defense response against insects and pathogens. The induction of PIs is known to be regulated by endogenous chemical factors including phytohormones. We studied the induction of barley chymotrypsin and trypsin inhibitory activities by aphid infestation, mechanical wounding, abscisic acid (ABA) and jasmonic acid (JA). Wounding experiments led to a minimal accumulation of PI activity (16% over controls) compared to that found in barley seedlings infested by aphids, where chymotrypsin inhibitor activity showed a two-fold increment. No systemic induction could be detected in healthy leaves of an infested or mechanically injured plant. Exogenous ABA applied on barley leaves increased the chymotrypsin inhibitory activity, while JA only increased trypsin inhibitory activity locally and systemically when applied exogenously. Our data suggest that two different mechanisms may be regulating the induction of these two types of inhibitors.     

Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree)

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.     

Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects.

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.     

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.     

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.     

Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from theobroma seeds on midgut protease activity of lepidopteran pest insects

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.     

Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.     

Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco

A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T₀ and T₁ plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.     

Momordica charantia trypsin inhibitor II inhibits growth and development of Helicoverpa armigera

Abstract  Bitter gourd ( Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs), which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera . In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-II, its cloning and expression as a recombinant protein using Pichia pastoris have been reported. Recombinant McTI-II inhibited bovine trypsin at 1: 1 molar ratio, as expected, but did not inhibit chymotrypsin or elastase. McTI-II also strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H. armigera larvae. The insect larvae fed with McTI-II-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding. Moreover, ingestion of McTI-II resulted in 23% mortality in the larval population. The strong antimetabolic activity of McTI-II toward H. armigera indicates its probable use in developing insect tolerance in susceptible plants.