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Dominique Michaud, Thierry C. Vrain and Hugh A. Daubeny

Transformation of plant genomes with cysteine proteinase inhibitor (cystatin) genes represents an attractive option for the biological control of insect pests. However, this strategy must be carefully considered, because the transgenic plant endogenous proteinases may represent potential target enzymes for the exogenous inhibitors produced. For example, we are considering the transformation of strawberry (Fragaria ×ananassa) with cystatin cDNA clones, to control the Coleoptera pest black vine weevil (BVW; Otiorynchus sulcatus). Electrophoretic analyses of adult BVW proteinases have revealed the involvement of at least five proteinase forms for protein digestion, and the major form was strongly inhibited by oryzacystatins (OCI and OCII), two cystatins isolated from rice seeds. A similar analysis of proteinases showed the existence of OC-sensitive proteinase activity in the leaves of strawberry, suggesting a possible risk of interference of the inhibitors in the transformed plants. In addition, the two rice inhibitors were rapidly hydrolyzed at 25C when incubated with proteinase extracts from either young, mature or senescent leaves. An efficient control of BVW by plant cystatin-expressing transgenic strawberry plants is therefore potentially possible, but the correct targeting of the inhibitors in the plant cells using appropriate signal peptides could be necessary.

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Dominique Michaud, Serge Overney, Binh Nguyen-Quoc and Serge Yelle

In the past few years, transformation of plant genomes with proteinase inhibitor (PI) genes has been proposed as an effective way to produce insect-tolerant plants. For such a control approach, however, biochemical studies are necessary to assess the effect of PIs on not only insect digestive proteinases (target enzymes) but also plant endogenous proteinases (nontarget enzymes). As an example, transformation of potato (Solanum tuberosum L.) with oryzacystatin (OC) genes, two cysteine PIs, was considered for controlling Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The use of electrophoretic approaches and standard assays showed that CPB uses at least 14 cysteine proteinases for protein digestion throughout its development. Proteinases of the same class were also detected in sprouting potato tuber extracts, suggesting a potential interference of cPIs in transgenic plants. While OCs inhibit a significant fraction of CPB digestive proteinases, no inactivation of potato proteinases was detected. This apparent absence of direct interference suggests the real potential of OCs for producing CPB-tolerant transgenic potato plants.

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Serge Overney, Dominique Michaud, Binh Nguyen-Quoc and Serge Yelle

In recent years, several studies have demonstrated the potential of proteinase inhibitors (PIs) for controlling insect pests. Used as a component of an integrated pest management program, such an approach must, however, be considered with care, given the potential risks of interference on other control approaches. In particular, the effect of PIs on digestive proteinases of beneficial insects must be determined. As an example, this study analyzed the effect of oryzacystatins (OCs), two cysteine PIs isolated from rice, on digestive proteinases of Perillus bioculatus, a predator of the Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), a major pest. Electrophoretic analyses showed the existence of several cysteine proteinase forms in the digestive tract of P. bioculatus. For each developmental stage, OCs dramatically inhibited proteolytic activity, showing an affinity between these inhibitors and the digestive proteinases of the predator. Despite their potential for controlling CPB, the two rice cystatins thus represent possible growth-suppressing compounds for the beneficial insect P. bioculatus. Work is currently under way to assess the compatibility of the two control approaches.

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Dominique Michaud, Thierry C. Vrain, David A. Raworth and Hugh A. Daubeny

In recent years, several studies have demonstrated the potential of proteinase inhibitors (PIs) for the control of various pests and pathogens. Used as a component of an integrated pest management program, such an approach must, however, be carefully considered, given the possible risks of interference with other control methods. For example, we are analyzing the effect of oryzacystatins (OCI and OCII), two cysteine PIs naturally occurring in rice grains, against digestive proteinases of Amblyseius californicus (AC), a native predator of the two-spotted spider mite (SM; Tetranychus urticae). Electrophoretic analyses have shown the existence in SM extracts of a major cysteine proteinase form strongly inhibited by OCI, indicating the potential of this inhibitor for SM control. However, similar analyses revealed a strong affinity between proteinases from AC extracts and OCs. Thus, despite their potential for SM control, plant cystatins may represent growth-suppressing compounds for AC. Work is currently underway to determine the usefulness of OCI-expressing transgenic plants for SM control, and to assess the compatibility of this control with an AC-based biological control strategy.