ENGINEERED ORYZACYSTATIN-I EXPRESSED IN TRANSGENIC HAIRY ROOTS CONFERS RESISTANCE TO GLOBODERA-PALLIDA

The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated. Inhibitory activities (K-i) of wild-type and variant forms of Oc-I against both papain and the C. elegans cysteine proteinase, gcp-1, were measured. For one variant, in which residue Asp86 was deleted (Oc-I Delta D86), the K-i was reduced by 13- to 14-fold. LD50 studies to test the effect of Oc-I and Oc-I Delta D86 against C. elegans showed the relative median potency of OcI Delta D86 to be 0.76 that of wild-type Oc-I. When expressed in tomato hairy roots both Oc-I and Oc-I Delta D86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc-I Delta D86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.