In vivo laboratory bioassays conducted in our laboratory have demonstratd that selected microbial and synthetic cysteine proteinase inhibitors significantly inhibited alfalfa weevil foliar feeding, pupation, adult emergence, and fecundity. Several plant lectins also were identified which significantly inhibited many of these same variables. Two cDNA clones of cysteine proteinase inhibitor genes from rice have been recloned into an E. coli expression vector for recombinant protein syntheses and their effects tested in vivo on alfalfa weevil growth and development and in vitro on alfalfa weevil midgut activity. These same genes have been reconstructed for expression in plant cells, cloned into transformation vectors carrying reporter and selectable marker genes and introduced into alfalfa explants via Agrobacterium-mediated gene transfer methods. Regenerated alfalfa transformants are currntly being vegetatively propagated and will be characterized for protein expression of the inhibitor genes by molecular techniques and in vivo laboratory insect bioassays.
Reports in the literature on other insect protein-inhibitor relationships suggest that insects utilize several classes of proteinases for digestion and that within a specific class there may be multiple forms controlled by different genes. In order to disrupt the digestive physiology of a specific insect it may be necessary to target multiple proteinases. Efforts are currently underway to identify multiple proteinase genes that may be involved in alfalfa weevil digestion so that these major proteinases can be targeted by inhibitors that are specific and potent. Towards this approach we have recently cloned a cysteine proteinase gene from the alfalfa weevil midgut and hope to identify additional cysteine proteinase genes. These genes will be used to express recombinant proteins for screening a library of novel cysteine proteinase inhibitors for selected variants with the highest binding affinity and insecticidal activity.