Protease inhibitors and the biochemical basis of insect resistance in Vigna unguiculata

Abstract

The cowpea (Vigna unguiculata) suffers severe losses (up to 70%) when stored due to infestation by the bruchid beetle Callosobruchus maculatus. A variety of cowpea resistant to bruchid attack had been described (IITA); the present work confirmed its resistance. The resistance was shown not to be of a physical nature, and thus seeds of both this resistant variety and susceptible varieties were screened for all possible toxins which were likely to form the biochemical basis of the resistance. The trypsin inhibitor was the only toxin detected. Using two different methods both for extraction and determination of the inhibitory activity, the trypsin inhibitor was found to be present in about twice the concentration in seeds of the resistant variety compared to the highest level found in susceptible varieties. The trypsin inhibitor was purified by affinity chromatography, and its toxicity towards larvae of Callosobruchus maculatus was demonstrated directly by feeding trials. Addition of the trypsin inhibitor, at the physiological concentration of the resistant variety, to the basic diet was found to be lethal, whereas addition at the physiological concentration found in susceptible varieties had no apparent effect. Further, addition of the inhibitor to the level of becoming resistant. Inactivation of the trypsin inhibitor removed its antimetabolic activity. The toxicity was also demonstrated in vitro by inhibition of larval proteolysis of synthetic and physiological substrates. A partial characterisation of the cowpea trypsin inhibitor was carried out. It was shown to have a molecular weight of approximately 17,000. Complex formation with trypsin could be directly demonstrated. The inhibitor as isolated was a mixture of several isoinhibitors, some of which were able to inhibit chymotrypsin as well as trypsin. A model for the subunit structure of the inhibitor is proposed.