title: Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida.
abstract: 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 (Ki) 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 deltaD86), the Ki 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 Oc-I 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 deltaD86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.
authors.affiliation: Centre for Plant Biochemistry and Biotechnology, University of Leeds, UK.
authors.forename: P E
authors.initials: PE
authors.lastname: Urwin
authors.forename: H J
authors.initials: HJ
authors.lastname: Atkinson
authors.forename: D A
authors.initials: DA
authors.lastname: Waller
authors.forename: M J
authors.initials: MJ
authors.lastname: McPherson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000254:growth & development; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D018551:Solanum lycopersicum / Q000235:genetics* / Q000276:immunology / Q000469:parasitology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009348:Nematoda / Q000254:growth & development*; D018517:Plant Roots / Q000378:metabolism*; D030821:Plants, Genetically Modified; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid
chemical_list: D015891:Cystatins; D017931:DNA Primers; D003546:Cysteine Endopeptidases