Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nM to 30 nM. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.Keywords: gut; Helicoverpa; inhibitor; insect; trypsin.Larvae of the lepidopteran insect species Helicoverpa are a pest in Asia, Australia and the Americas. They cause yield losses on many important crops, like cotton, chickpea, corn, and tomato. For instance, of the total cotton area in China (4.7 million hectares), 30% was lost to H. armigera in the mid nineties [1]. Chemical control of Helicoverpa insects is often not effective, as they are notorious for development of resistance to chemicals such as DDT, organophosphates and pyrethroids [2].One form of natural defence of plants against insects is mediated by protease inhibitors [3]. The inhibitors are thought to have coevolved with insect herbivory, and to function by blocking the digestive proteases in the larval gut, thereby limiting the release of amino acids from food protein. As a consequence, the larvae are arrested in development, and eventually die. Genes encoding protease inhibitors have been used to produce resistant transgenic plants as a crop-protection strategy. This has met with initial success [4-6], but disappointing results have been reported for Helicoverpa spp., and a few other pest insects. Although several groups have shown that a major part of Helicoverpa gut protease activity can be blocked by a number of inhibitors [7,8], even the most efficient inhibitor (soybean Kunitz trypsin inhibitor, SKTI), which inhibits 95% of trypsin activity in gut extracts, does not affect the larval development of this insect on artificial diet [9] or transgenic plants [8].The lack of effect on larval development is caused by the adaptation of Helicoverpa spp. to protease inhibitors, which is mediated by their ability to alter the complement of proteolytic activity in their gut. In response to inhibitor ingestion, the arsenal of...

PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs.

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.

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 401 60 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine L L-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.z 2000 Federation of European Biochemical Societies.

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