Abstract:
Cysteine protease inhibitors (cystatins) are expressed in plants in response to wounding and insect herbivory and they form part of the native host-plant defence system. Cysteine proteases are enzymes important in the break down of dietary proteins mainly in the mid gut of coleopteran insects such as the banana weevil. The inhibition of these proteases has a direct effect on the digestive activity of the insect resulting in protein deficiency. This significantly affects insect development and survival. Based on these observations, strategies have been designed involving expression of cysteine protease inhibitors for the transgenic control of insect pests of several crop plants. For this study, it was hypothesized that the major proteases in banana weevil are cysteine proteases and can be effectively targeted by plant cystatins. It was further hypothesised that since plant cystatins are defense related, certain amino acid residues may have undergone positive selection. This provides an opportunity to increase their inhibitory potential to the weevil gut proteases via protein engineering. To prove the hypotheses, both in-vitro and in-vivo assays were set up thus allowing us to demonstrate the presence of cysteine type proteases banana weevil as well as the effect of cystatins on the weevil proteases and early development. Initial in-vitro experiments were able to characterize the proteolytic activity of the banana weevil gut proteases, which are mostly of the cysteine type, and in particular cathepsin B and L like. Two recombinant phytocystatins were further successfully produced using a 6xHis-tagged affinity chromatogephy system in Escherichia coli bacteria. The recombinant phytocystatins were used in a newly developed vacuum infiltration assay system using banana stems. Young weevil larvae were allowed to develop on phytocystatin-treated stems for up to 10 days. They had a 60% reduction in body weight and rate of growth compared to those that grew in untreated stems. By carrying out sitedirected mutagenesis to improve the inhibition efficiency of a model papaya cystatin, more than 8 amino acid residues were found to be subjected to positive selection. Mutation of amino acids yielded improved the inhibition potential of papaya cystatin against the model cysteine protease papain. Increased inhibition was greatest when amino acids were changed in the highly variable regions of the amino acid sequence very closely to the conserved regions. This study has been able to show for the first time that banana weevils use cysteine protease as major protein hydrolysis enzymes and that these can be effectively targeted by plant cystatins. It has also created novel phytocystatins using engineering of single amino acid sites following an evolutionary approach to modulate them for improved activity and targeting specific proteases.
