Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.     

Crystal structure reveals basis for the inhibitor resistance of human brain trypsin

Severe neurodegradative brain diseases, like Alzheimer, are tightly linked with proteolytic activity in the human brain. Proteinases expressed in the brain, such as human trypsin IV, are likely to be involved in the pathomechanism of these diseases. The observation of amyloid formed in the brain of transgenic mice expressing human trypsin IV supports this hypothesis. Human trypsin IV is also resistant towards all studied naturally occurring polypeptide inhibitors. It has been postulated that the substitution of Gly193 to arginine is responsible for this inhibitor resistance. Here we report the X-ray structure of human trypsin IV in complex with the inhibitor benzamidine at 1.7 A resolution. The overall fold of human trypsin IV is similar to human trypsin I, with a root-mean square deviation of only 0.5 A for all C(alpha) positions. The crystal structure reveals the orientation of the side-chain of Arg193, which occupies an extended conformation and fills the S2' subsite. An analysis of surface electrostatic potentials shows an unusually strong clustering of positive charges around the primary specificity pocket, to which the side-chain of Arg193 also contributes. These unique features of the crystal structure provide a structural basis for the enhanced inhibitor resistance, and enhanced substrate restriction, of human trypsin IV.     

Changes in the activity of proteases and trypsin inhibitors in wheat leaves in the initial period of cold hardening and subsequent dehardening

The dynamics of amidase, cysteine protease, and trypsin inhibitor activities were studied in the leaves of wheat (Triticum aestivum L.) seedlings grown under controlled conditions (25°C, illuminance 10 kLx, 14-h photoperiod) and subjected to cold hardening (5°C, 10 kLx, 14-h photoperiod). Changes in the activity of amidases and cysteine proteases proved to precede an increase in cold resistance during cold hardening and a decrease in cold resistance after the end of cold hardening. The activity of trypsin inhibitors changed only during cold hardening. It is suggested that amidases, cysteine proteases, and trypsin inhibitors are involved in the cold adaptation of plants.     

Antifungal Activity of a Bowman–Birk-type Trypsin Inhibitor from Wheat Kernel

A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC₅₀) values ranging from 111.7 to above 500 μg/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

A possible evolutionary relationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretory trypsin inhibitor (Kazal)

The amino acid sequence of the carboxyl-terminal half of barley trypsin inhibitor was found to be significantly similar to the whole sequence of bovine pancreatic secretory trypsin inhibitor (Kazal). Kazal type inhibitors and related proteins are known for the extraordinary mode of divergence among animals, and the present observation extends this to a plant for the first time. The present observation together with our previous finding of sequence homology between barley trypsin inhibitor and wheat alpha-amylase inhibitor (Odani, S., Koide, T., & Ono, T. (1982) FEBS Lett. 141, 279-282) suggest an unusual evolutionary relationship between cereal enzyme inhibitors and animal proteinase inhibitors of the Kazal type.     

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye)

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex (2:1) at 2.3 a resolution

The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 A resolution to a final R-factor of 0.177. A distance of 37.2 A between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten beta-strands and the loops connecting these strands but it lacks alpha-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel beta-strands, beta 1/beta 2 and beta 6/beta 7. Strands beta 1 and beta 6 are in direct contact with trypsin molecules and form stable triple stranded beta-sheet structures via hydrogen bonds.     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Production and properties of recombinant glutenin-hydrolyzing proteinases from Eurygaster integriceps Put.

cDNAs coding for a mature form of glutenin-hydrolyzing trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes.     

Response of corn plants to the effect of salicylic acid and Fusarium moniliforme

Effect of salicylic acid and Fusarium moniliforme on trypsin inhibitor activity, lectine activity, lectine carbohydrate specificity, and salicylic acid content in sprouted maize was studied. Changes in trypsin inhibitor activity, lectine activity, and content of endogenous salicylic acid during action of exogenous salicylic acid or pathogen were shown to depend on resistance of maize lines to fusariosis pathogen. Salicylic acid was proposed to take part in induction of trypsin and lectine inhibitors. Trypsin and lectine inhibitors are important in formation of sprouted maize resistance to abiotic and biotic factors.     

The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein

The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.     

Constitutive expression of a cowpea trypsin inhibitor gene,CpTi, in transgenic rice plants confers resistance to two major rice insect pests

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.     

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.