Plant Physiol. (1998) 118: 997-1004

Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants¹

Francesca De Leo, Michel A. Bonadé-Bottino, Luigi R. Ceci, Raffaele Gallerani, and Lise Jouanin^*

Dipartimento di Biochimica e Biologia Moleculare, Università di Bari, Via Orabona 4, 70126 Bari, Italy (F.D.L., R.G.); Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France (M.A.B.-B., L.J.); and Centro di Studio sui Mitocondri e Metabolismo Energetico-Consiglio Nazionale delle Richerche, Sezione di Trani, Via Corato 17, 70059 Trani, Italy (L.R.C.)

	ABSTRACT

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This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.

	INTRODUCTION

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PIs are widely spread throughout the plant kingdom. They are known to be involved in several physiological processes, such as reserve control and defense against pathogens and pests (Koiwa et al., 1997). In the latter case, PIs have been shown to be developmentally expressed in seeds and reserve organs (Birk, 1994; Koiwa et al., 1997) or induced by wounding in leaves (Schaller and Ryan, 1995). The natural protective role of PIs against phytophageous insects and the availability of PI-encoding sequences encouraged the development of pest-resistance programs based on PI expression in transgenic plants (Ryan, 1990).

Despite several reports of successful protection of plants and trees against phytophageous insects from several taxonomic orders, mainly Lepidoptera (Jongsma and Bolter, 1997; Gatehouse, 1998; Jouanin et al., 1998; Schuler et al., 1998), defense strategies based on PI expression in plants have not resulted in any commercial application so far. This relates to two distinct problems: (a) the pests' capacity to react to PI consumption, and (b) the PI-expression levels in transgenic plants.

The first point is exemplified by the fact that PIs shown to be potent inhibitors of insect gut proteinases in in vitro assays failed to produce any deleterious effect when fed to larvae (Baker et al., 1984; Purcell et al., 1992). Recent data provide some insights into the insect pests' ability to overcome the potential deleterious effects caused by PI consumption. Several mechanisms were reported: the inactivation of PI by insensitive proteinases (Jongsma and Bolter, 1997; Michaud, 1997) and the synthesis of novel proteinases insensitive to the PI ingested (Jongsma et al., 1996; Jongsma and Bolter, 1997). To counter such resistance mechanisms, several strategies have been proposed for the design of more efficient pest-resistant transgenic plants: the expression in the same plant of several genes encoding entomopathogenic molecules (PI and other types of molecules such as Bacillus thuringiensis toxins) and/or the use of engineered inhibitors with increased specificity or a larger spectrum (Jongsma et al., 1996; Jongsma and Bolter, 1997; Michaud 1997; Reeck et al., 1997).

In comparison with the pests' adaptation capacities, little attention has been given to potential problems linked to the level of expression of PI genes in transgenic plants. Reports have shown that to cause deleterious effects on pests, PI-expression levels have to be relatively high, on the order of 1% of total soluble protein, as previously shown in bioassays of artificial diets (Hilder et al., 1987; Leplé et al., 1995). Nevertheless, to our knowledge, no attempt has been made to estimate the possible occurrence of adverse effects linked to inadequate PI-expression levels in transgenic plants. In view of a possible field release of transgenic plants expressing PI, this point appears to be of crucial importance.

To test the possibility of the occurrence of negative effects linked to PI-expression levels, tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis L. (Heynh.) ecotype Wassilewskija transgenic plants constitutively expressing MTI-2 were obtained. MTI-2 was isolated from seeds of white mustard (Menegatti et al., 1992) and is related (70% amino acid homology) to the rapeseed trypsin inhibitor (Ceciliani et al., 1994). Both PIs have primary structure characteristics that are uncommon to PI families described so far (Reeck et al., 1997). In mustard, MTI-2 exhibits expression patterns (Ceci et al., 1995) similar to that of PIs known to be part of plant-defense mechanisms in potato (Suh et al., 1991) and tomato (Wingate and Ryan, 1991). Therefore, MTI-2 is thought to be a good candidate for enhancing pest resistance in plants. In this paper we report the effect of consumption by larvae of a lepidopteran pest, the Egyptian cotton worm (Spodoptera littoralis Boisd.), of leaves of tobacco and Arabidopsis plants expressing both low and high levels of MTI-2.

	MATERIALS AND METHODS

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Construction of Plasmid Vector pKY-MTI-2

View larger version (8K): [in this window] [in a new window]   Figure 1. Structure of the pKY-MTI-2 T-region. Pnos, Tnos, Nopaline synthase promoter and terminator, respectively. nptII, Neomycin phosphotransferase-coding sequence; mti-2, MTI-2-coding sequence; P35S2, 35S RNA promoter with a doubled enhancer; trbcS, pea Rubisco terminator; LB, RB, left and right border sequences, respectively. The arrows indicate the direction of transcription of the mti-2 and nptII genes.

Plant Transformation and Regeneration

Plant Preparation

MTI-2 Expression in Leaves of Transgenic Plants

Feeding Bioassays

Gut pH Determination

Proteinase Activity in S. littoralis

Detection of Digestive Proteinase in S. littoralis by Two-Step Gelatin/PAGE

Leaf-Surface Consumption Analysis

Statistics

	RESULTS

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Plant Transformation and in Vitro Analysis of MTI-2 Expression

By comparing on activity gels the trypsin inhibition by leaf extracts of MTI-2-expressing plants with the trypsin inhibition by soybean trypsin inhibitor, Kunitz type, we estimated that the high-expression line (2T) expressed MTI-2 at about 1.6% of leaf-soluble proteins, whereas in the low-expression line (4T) MTI-2 accounted for 0.5% of the soluble proteins. All Arabidopsis lines expressed MTI-2 at a comparable level, similar to that observed in tobacco line 2T (Table I). Therefore, only one single-copy homozygous line, noted 7A, was selected. In both tobacco and Arabidopsis, northern-blot analysis gave results concurrent with azocasein tests (results not shown). Figure 2 shows a comparison of -bovine trypsin inhibitory activity of the selected transgenic lines. In Arabidopsis, a weak trypsin inhibition activity was observed in line CA, at a molecular size similar to that of MTI-2. This may be related to the presence in the Arabidopsis genome of a PI gene (accession no. AC002355) with high sequence homology to MTI-2. All selected lines were subsequently used in S. littoralis larvae-feeding bioassays.

View larger version (73K): [in this window] [in a new window]   Figure 2. Visualization of MTI-2 in transgenic plants by gelatin/PAGE. Plant extracts were separated in acrylamide gels containing gelatin. Gelatin was subsequently degraded by incubation of the gel in a trypsin-containing solution. Trypsin inhibition by 10 µg (tobacco) and 5 µg (Arabidopsis) of soluble proteins from leaf extract was visualized by the undegraded blue-stained gelatin located where the PI initially migrated.

Bioassays on S. littoralis Larvae

View larger version (24K): [in this window] [in a new window]   Figure 3. Comparison of weight of S. littoralis larvae fed on transgenic plant leaves for 10 d. A and B, Weight distribution in larvae fed on tobacco (A) or Arabidopsis (B); C, larvae grown on tobacco lines 2T, 4T, and CT. Each larvae shown here had a weight nearly equal to the mean weight observed in the insect pool to which it belonged.

To assess potential effects of MTI-2 consumption on the gut proteinase pool in S. littoralis larvae, proteinase activity was first analyzed in fourth-instar larvae grown on control plants. Activity profiles showed a peak of activity at pH 11.0 (Fig. 4), which is in agreement with previously published data for this insect (Ishaaya et al., 1971). This result is in accordance with the mean pH value observed for gut homogenate, i.e. pH 9.3. The overall activity showed a high sensitivity to the trypsin/chymotrypsin inhibitor BBI (Fig. 4), with 90% of activity inhibited at the optimum pH (Fig. 5A). Proteinase activity insensitive to BBI is likely to include elastase-like proteinase(s), since degradation of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, shown to be a substrate for gut chymotrypsin and elastase-like proteinase in other lepidopteran pests (Johnston et al., 1995), was only partly inhibited by the specific chymotrypsin inhibitor N-tosyl-L-Phe chloromethyl ketone (result not shown). E64, a potent inhibitor of Cys proteinases, had no significant effect on BBI-insensitive proteolytic activity (Fig. 4). The proteolytic complex was found to include at least four proteinases active at pH 11.0 (Fig. 5B). These proteinases were completely inhibited by BBI (result not shown), whereas protein extract from MTI-2-expressing plants inhibited all but P2 proteinase, which showed reduced activity (Fig. 5C). Overall, the digestive proteolytic system we observed in S. littoralis is consistent with previously published data for Noctuidae (Christeller et al., 1992), which had a proteinase activity that relied mainly on Ser proteinases, with a major component being trypsin-like proteinases (although chymotrypsin- and elastase-like proteinases are also present). The proteinase P2, which is partly inhibited by MTI-2, may account for part of this non-trypsin-like activity.

View larger version (15K): [in this window] [in a new window]   Figure 4. Determination of digestive proteinase activity in S. littoralis gut. Gut proteinase activity was monitored using azoalbumin, a general proteinase substrate, at different pHs. The activity was measured by the amount of liberated oligo- and polypeptides by reading A340. The activity was monitored without (basal) or with PIs: 0.1 mg/mL of the trypsin/chymotrypsin BBI or 0.1 mg/mL of BBI and 0.1 mM of the Cys PI E64 (BBI + E64).

View larger version (34K): [in this window] [in a new window]   Figure 5. Comparison of digestive proteinase activity (pH 11.0) of S. littoralis larvae fed on transgenic tobacco plants for 10 d. Gut extracts were labeled according to the transgenic plants they fed on. A, Activity monitored without (basal) or with BBI at 0.1 mg/mL (+BBI). Each point is the mean of two repetitions for at least six extracts. The confidence interval was computed at 10%. B and C, Gelatin/PAGE assays of gut protein extracts (5 µg of protein) incubated with protein-leaf extract (1 µg/µL) from the control (B) or the MTI-2-expressing plant line 2T (C). Proteinases present in control larvae are denoted P1 to P4, whereas proteinases induced in larvae fed line 2T tobacco plants are denoted N1 to N6.

Consumption of leaves from tobacco plants expressing low levels of MTI-2 resulted in an increase in total proteinase activity (Fig. 5A). This higher activity stemmed from an increased expression of proteinases already present in control insects (Fig. 5, B and C). The sensitivity of these newly induced proteinases to BBI (result not shown) and MTI-2 (Fig. 5C) appeared to be unchanged when compared with proteinases extracted from control larvae. On the contrary, the consumption of tobacco expressing high levels of MTI-2 resulted in a 33% decrease in the proteolytic activity at pH 11.0 (Fig. 5A), although six new proteinases appeared on the proteolytic profile (Fig. 5B). These new proteinases were completely inhibited by BBI (result not shown) and MTI-2 (Fig. 5C). On activity gels, there was no evidence for an increase in proteinase P3 (which is incompletely inhibited by MTI-2) activity compared with control larvae. It is interesting that, whatever the level of expression of MTI-2 in leaves, the amount of proteinase activity insensitive to BBI did not change significantly (Fig. 5A).

	DISCUSSION

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This work illustrates the risks associated with a strategy for pest control based on PI expression in transgenic plants. It concentrates on the occurrence of unexpected adverse effects as the result of inadequate PI expression. A variation of PI-expression levels within the range previously shown to provide insect resistance to transgenic plants (Hilder et al., 1987; McManus et al., 1994; Leplé et al., 1995; Duan et al., 1996; Xu et al., 1996; Gatehouse et al., 1997; Yeh et al., 1997) resulted in dramatic changes in the reaction of S. littoralis larvae to transgenic leaf consumption. When the trypsin PI MTI-2 was expressed at the highest level in tobacco and Arabidopsis transgenic plants, deleterious effects were observed on insect larvae, together with a reduction of leaf damage, thus providing effective pest resistance to the plant. These results are in agreement with those obtained by Yeh et al. (1997) on the closely related pest, Spodoptera litura F, when expressing a sweet potato trypsin inhibitor in tobacco. On the contrary, when MTI-2 was expressed at lower levels in tobacco plants, larvae developed faster, were bigger than on control plants, and caused more damage to the leaves. These results were related to physiological changes in the proteolytic profile of the digestive tract of S. littoralis larvae.

The growth-enhancing effect of PI-containing leaves has been previously reported in a few different studies. The Noctuidae Thysanoplusia orichalcea F., closely related to S. littoralis, showed an increased growth when fed tobacco expressing a chymotrypsin inhibitor inconsistently (McManus et al., 1994). The cabbage seed weevil (Coleoptera Psylliodes chrysocephala L.), raised on oilseed rape expressing the Cys PI OCI, exhibited an increased weight compared with controls, together with a doubling of both OCI-sensitive and -insensitive preexisting proteinases (Girard et al., 1998c). Similarly, our results show that the consumption by S. littoralis of leaves expressing MTI-2 at the lowest level induced an increase in preexisting proteinase expression in gut, although not as large as found with P. chrysocephala. The mechanisms underlying this increase in proteinase production and linking it to an increase in leaf consumption are still unknown. Proteinase overproduction associated with a reduced growth has often been reported when insects were fed diets containing PI (Jongsma and Bolter, 1997). Broadway and Duffey (1986) proposed that PI-induced deleterious effects are caused by stimulating proteinase overproduction aimed at compensating the inhibition of a proteolytic activity, thus inducing a shortage of some amino acids. Such a compensation mechanism may explain results obtained from tobacco plants expressing low levels of MTI-2, even though such a level of PI expression failed to induce deleterious effects on S. littoralis. The increase in leaf-surface consumption observed on low MTI-2-expressing plants may be a consequence of the decrease in the diet's quality due to the presence of MTI-2 and/or to the increase in gut proteolytic capacity of the larvae. Global proteinase activity enhancement could provide sufficient MTI-2-insensitive proteinases to counter, at least partly, the effect of the amount of MTI-2 present in the larvae's diet, e.g. by degrading this PI, as recently reported in other insects (Girard et al., 1998b; Giri et al., 1998).

The proteolytic activity observed in larvae fed leaves expressing high levels of MTI-2 differed greatly from that observed in larvae fed leaves with low MTI-2-expression levels. The 30% decrease in overall proteinase activity, compared with the control, was shown to coincide with the appearance of new proteinases. The induction of new forms of proteinase after feeding on a PI-containing diet has been previously described in other insects (Broadway, 1996; Jongsma and Bolter, 1997). Newly induced proteinases have been shown to exhibit a lower sensitivity toward the PI consumed by the insect. In S. littoralis we observed that all of the newly induced proteinases were still sensitive to MTI-2, suggesting that the deleterious effects observed on growth and mortality could be linked to a shortage of available amino acids because of extensive proteinase synthesis. Leaf damage was reduced the same way as overall proteinase activity, suggesting that the consumed leaf area is at least partly determined by the proteolytic activity in the gut.

The dramatic differences observed in larvae fed leaves expressing high or low levels of MTI-2 suggest the occurrence of a sensitivity threshold in S. littoralis toward MTI-2. This threshold represents the minimum concentration of MTI-2 in the larvae's diet necessary to induce deleterious effects. Below this threshold value, larvae are able to overcome the inhibition caused by PI ingestion by overexpressing digestive proteinases. Above this threshold, PI ingestion induces strong adaptive reactions, such as the production of new proteinases, mobilizing enough of the larvae resources to impair its growth. If the threshold is not reached, adverse effects such as damage enhancement and/or increased pest growth can occur.

This work addresses questions that must be considered on a case-by-case basis with regard to two different parameters: the target pest and the in planta expression characteristics. On the one hand, the sensitivity threshold for a same PI is likely to differ from insect to insect, since different insects have been shown to exhibit various sensitivities to a given PI and single insects have been shown to exhibit different sensitivities to various PIs (Larocque and Houseman, 1990; Christeller et al., 1992; McManus et al., 1994). The design of a pest-defense strategy for a crop using a particular PI is therefore dependent on the assessment of the sensitivity threshold of the target pest toward the chosen PI. In addition, care must be taken for the deployment of PI transgenic plants in the field, since different populations of the insect pests can exhibit different sensitivity levels to a same PI, as reported recently (Girard et al., 1998a). However, the stability of expression is paramount to the successful use of PI to protect plants against attack by insect pests. Aside from the problem of the choice of an adequate promoter, the development of a successful defense strategy against a given pest, based on PI expression in planta, must take into account potential variations in PI-expression levels in a plant (as a result of age, physiological status, etc.) and between plants of the same line (Elkind et al., 1995) to ensure that the sensitivity threshold of this pest for the PI is always reached. With regard to this requirement, the use of PI presenting a high affinity to an insect digestive proteinase is highly desirable, as lower PI-expression levels could be sufficient to impair development. Strategies aimed at improving the affinity of a PI for a target pest's major proteinase before its expression in plants (Jongsma et al., 1996) should therefore receive special attention.

	FOOTNOTES

   Received April 29, 1998; accepted August 17, 1998.

	ABBREVIATIONS

Abbreviations: 2T, MTI-2-expressing tobacco line 2. 4T, MTI-2-expressing tobacco line 4. 7A, Arabidopsis MTI-2-expressing line 7. BBI, soybean Bowman Birk Inhibitor. CA, Arabidopsis control line. CT, tobacco control line. E64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane. MTI-2, mustard trypsin inhibitor 2. PI, proteinase inhibitor.

	ACKNOWLEDGMENTS

We thank Dr. J. Chaufaux (Station de lutte biologique, Institut National de la Recherche Agronomique, La Minière, 78285 Guyancourt cedex, France) for providing S. littoralis larvae and Dr. M. LeMétayer (Laboratoire de Neurobiologie Comparée des Invertébrés, Institut National de la Recherche Agronomique, BP23, 91440 Bures sur Yvette, France) for technical assistance during image-analysis sessions. The authors are also grateful to Dr. M. Giband for critical reading of the manuscript.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Baker J, Woo S, Mullen M (1984) Distribution of proteinases and carbohydrases in the midgut of larvae of the sweet potato weevil Cylas elegantulus and response of proteinases to inhibitors from sweet potato. Entomol Exp Appl 36: 97-105

Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Ser III Sci Vie 316: 1194-1199

Birk, Y (1994) Protein proteinase inhibitors in legume seeds-overview. Arch Latinoam Nutr 44: 26S-30S

Broadway RM (1996) Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 32: 39-53

Broadway R, Duffey S (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis virescens and Spodoptera exigua. J Insect Physiol 32: 827-833 [CrossRef]

Ceci LR, Spoto N, de Virgilio M, Gallerani R (1995) The gene coding for the mustard trypsin inhibitor-2 is discontinuous and wound-inducible. FEBS Lett 364: 179-181 [CrossRef][Medline]

Ceciliani F, Bortolotti F, Menegatti E, Ronchi S, Ascenzi P, Palmieri S (1994) Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (Brassica napus) seeds. FEBS Lett 342: 221-224 [CrossRef][Medline]

Christeller JT, Laing WA, Markwick NP, Burgess EPJ (1992) Midgut protease activities in 12 phytophageous lepidopteran larvae: dietary and protease inhibitor interactions. Insect Biochem Mol Biol 22: 735-746 [CrossRef]

Duan X, Li X, Xue Q, Abo-El-Saad M, Xu D, Wu R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnol 14: 494-498 [CrossRef][ISI][Medline]

Elkind Y, Nir B, Nadler-Hassar T (1995) Quantitative analysis of the transgene variability among primary tobacco transformants. Transgenic Res 4: 30-38

Gatehouse AMR (1998) Biotechnological application of plant genes in the production of insect resistant crops. In SL Clement, SS Quinsenberry, eds, Global Plant Genetic Resources for Insect Resistant Crops, Chap 15. CRC Press, Boca Raton, FL (in press)

Gatehouse AMR, Davison GM, Newell CA, Merryweather A, Hamilton WDO, Burgess EPJ, Gilbert RJC, Gatehouse JA (1997) Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials. Mol Breeding 3: 49-63

Girard C, Bonadé-Bottino M, Pham-Delègue MH, Jouanin L (1998a) Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I. J Insect Physiol 44: 569-577 [CrossRef]

Girard C, Le Métayer M, Bonadé-Bottino M, Pham-Delègue MH, Jouanin L (1998b) High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Mol Biol 28: 229-237 [CrossRef][ISI][Medline]

Girard C, Le Métayer M, Zaccomer B, Bartlet E, William I, Bonadé-Bottino M, Pham-Delègue MH, Jouanin L (1998c) Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor. J Insect Physiol 44: 263-270 [Medline]

Giri AP, Harsulkar AM, Deshpande VV, Sainani MN, Gupta VS, Ranjekar PK (1998) Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 116: 393-401 [Abstract/Free Full Text]

Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 300: 160-163

Ishaaya I, Moore I, Joseph D (1971) Protease and amylase activity in larvae of the Egyptian cotton worm, Spodoptera littoralis. J Insect Physiol 17: 945-953

Johnston KA, Lee MJ, Brough C, Hilder VA, Gatehouse AMR, Gatehouse J (1995) Protease activities in the larval midgut of Heliothis virescens: evidence for trypsin and chymotrypsin-like enzymes. Insect Biochem Mol Biol 25: 375-383

Jongsma MA, Bolter C (1997) The adaptation of insect to plant protease inhibitors. J Insect Physiol 43: 885-896 [CrossRef][ISI][Medline]

Jongsma MA, Stiekema WJ, Bosch D (1996) Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 14: 331-333 [CrossRef]

Jouanin L, Bonadé-Bottino M, Girard C, Morrot G, Giband M (1998) Transgenic plants for insect resistance. Plant Sci 131: 1-11 [CrossRef]

Koiwa H, Bressan RA, Hasegawa PM (1997) Regulation of protease inhibitors and plant defense. Trends Plant Sci 2: 379-384 [CrossRef][ISI]

Larocque AM, Houseman JG (1990) Effect of ingested soybean, ovomucoid and corn protease inhibitors on digestive processes of the European corn borer, Ostrinia nubilalis (Lepidoptera: pyralydae). J Insect Physiol 36: 691-697

Leplé JC, Bonadé-Bottino M, Augustin S, Pilate G, Dumanois G, Le Tan VD, Delplanque A, Cornu D, Jouanin L (1995) Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1: 319-328 [CrossRef]

Maiti IB, Murphy JF, Shaw AG (1993) Plants that express a potyvirus VPg-proteinase gene are resistant to virus infection. Proc Natl Acad Sci USA 90: 6110-6114 [Abstract/Free Full Text]

Mathis NL, Hinchee MAW (1994) Agrobacterium inoculation techniques for plant tissues. In SB Gelvin, RA Schilperoort, eds, Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-9

McManus MT, White DWR, McGregor PG (1994) Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 3: 50-58

Menegatti E, Tedeschi G, Ronchi S, Bortolotti F, Ascenzi P, Thomas RM, Bolognesi M, Palmieri S (1992) Purification, inhibitory properties and amino acid sequence of a new serine proteinase inhibitor from white mustard (Sinapis alba L.) seeds. FEBS Lett 301: 10-14 [CrossRef][Medline]

Michaud D (1997) Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 15: 4-6

Michaud D (1998) Gel electrophoresis of proteolytic enzymes. Anal Chim Acta (in press)

Michaud D, Faye L, Yelle S (1993) Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrilamide gels and class-specific proteinase inhibitors. Electrophoresis 14: 94-98 [CrossRef][ISI][Medline]

Purcell JP, Greenplate JT, Sammons RD (1992) Examination of midgut luminal proteinase activities in six economically important insects. Insect Biochem Mol Biol 22: 41-47

Reeck GR, Kramer KJ, Baker JE, Kanost MR, Fabrick JA, Behnke CA (1997) Proteinase inhibitors and resistance of transgenic plants to insects. In N Carozzi, M Koziel, eds, Advances in Insect Control. The Role of Transgenic Plants. Taylor and Francis, London, pp 157-183.

Ryan CA (1990) genes for improving defences against insects and pathogens Annu Rev Phytopathol 28: 425-449

Schaller A, Ryan CA (1995) Systemina polypeptide defense signal in plants. Bioessays 18: 27-33

Schuler TH, Poppy GM, Kerry BR, Denholm I (1998) Insect-resistant transgenic plants. Trends Biochem Sci 16: 168-175

Suh SG, Stiekema WJ, Hannapel DJ (1991) Proteinase-inhibitor activity and wound-inducible gene expression of the 22-kDa potato-tuber proteins. Planta 184: 423-430

Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) EMBO J 2: 411-417 [ISI][Medline]

Wingate VPM, Ryan CA (1991) Uniquely regulated proteinase inhibitor I gene in a wild tomato species. Inhibitor I family gene is wound-inducible in leaves and developmentally regulated in fruit. Plant Physiol 97: 496-501 [Abstract/Free Full Text]

Xu D, Xue Q, McElroy D, Mawal Y, Hilder VA, Wu R (1996) Constitutive expression of a cowpea trypsin inhibitor gene CpTI in transgenic rice plants confers resistance to two major insect pests. Mol Breeding 2: 167-173

Yeh KW, Lin MI, Tuan SJ, Chen YM, Lin CY, Kao SS (1997) Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep 16: 696-699 [CrossRef]

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