A barley cysteine-proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants.

Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 → G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzus persicae and Acyrthosiphon pisum. All the cystatins, except HvCPI-7, HvCPI-10 and HvCPI-12, inhibited in vitro the activity of cathepsin L- and/or B-like proteinases, with HvCPI-6 being the most effective inhibitor for both aphid species. When administered in artificial diets, HvCPI-6 was toxic to A. pisum nymphs (LC(50) = 150 μg/ml), whereas no significant mortality was observed on M. persicae nymphs up to 1000 μg/ml. The effects of HvCPI-6 ingestion on A. pisum were correlated with a decrease of cathepsin B- and L-like proteinase activities. In the case of M. persicae, there was an increase of these proteolytic activities, but also of the aminopeptidase-like activity, suggesting that this species is regulating both target and insensitive enzymes to overcome the effects of the cystatin. To further analyze the potential of barley cystatins as insecticidal proteins against aphids, Arabidopsis plants expressing HvCPI-6 were tested against M. persicae. For A. pisum, which does not feed on Arabidopsis, a combined diet-Vicia faba plant bioassay was performed. A significant delay in the development time to reach the adult stage was observed in both species. The present study demonstrates the potential of barley cystatins to interfere with the performance of two aphid species.