Aphids constitute an important group of insect pests, and though, a limited number of genes encoding aphid-efficient toxins are available. The two pioneering gene families used to control the major insect pests have no efficient aphid-specific representatives: no Bacillus thuringiensis toxin with adequate aphid toxicity has been described, and no protease inhibitor (PI) with convincing aphid toxicity have been used yet in transgenic plants for aphid control. Since aphids feed on a low-protein diet (phloem sap), and do not rely on protein digestion for their nitrogen nutrition, the general absence of activity of PIs in early screening tests was not a surprise. However, several results obtained in vitro highlighted the interest of some members of this wide peptide class: First, one PI member of the plant cysteine protease inhibitors, oryzacystatin (gene OC I), showed a low but significant growth inhibition activity on many aphid species tested in vitro. Furthermore, transgenic oilseed rape plants expressing OC I in their phloem sap affected similarly the larval growth of Myzus persicae, and induced significant reductions in aphid fecundity. Finally, tissue and enzyme targets of OC I were successfully identified by immunolocalization and enzymatic assays on OC I-fed aphids; cysteine-type proteases were shown to form the major endoproteolytic system in the aphid digestive tract, and were analyzed. Although the digestive function of these proteases is still under question, the aphid mesenteron is definitely not devoid of protease activity. Second, a pea seed Bowman-Birk trypsin/chymotrypsin inhibitor was shown to induce significant in vitro toxicity towards the pea aphid Acyrthosiphon pisum. Testing isoforms and species variants of this bi-functional inhibitor indicated that toxicity was probably harbored by the anti-chymotrypsin active site ; this was confirmed by testing the effect of synthetic active-site peptides, which demonstrated that an active 15 amino acid anti-chymotrypsin loop was responsible for the acute toxicity observed on A. pisum. In conclusion, our work points out to the interest of some protease inhibitors for controlling insect groups initially not ideally suited to this type of activity. These results could be extended to other insect groups feeding on low-protein diets, such as other phloem or xylem-feeding Hemiptera.

Index terms: Aphididae, Myzus persicae, Acyrthosiphon pisum, Protease inhibitors, Bowman-Birk chymotrypsin inhibitor, Oryzacystatin, Cysteine proteases, Transgenic plants, Host-Plant Resistance.

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