Characterization of oxalate oxidase : a plant enzyme capable of degrading the major phytotoxin produced by Sclerotinia sclerotiorum

Abstract

Sclerotinia sclerotiorum is a phytopathogenic fungus which infects a wide range of plant species including some major crops such as sunflower and rape seed. In sunflower, the fungus infects via the roots causing root rot, basal stem canker, and wilting of the plant or under certain environmental conditions, airbone ascospores are produced which may incite head rot. During infection, the fungus produces high level of a necrosis phytotoxin identified as oxalic acid. The role of oxalic acid in the pathogenicity process is still unclear. However, oxalic acid could act synergically with pectolytic enzymes by lowering the pH of the infected tissue from 6.8 to 4 and by chelating divalent cations (Ca2+) which renders the Ca-pectate cell wall complex more susceptible to hydrolysis. The degradation of oxalic acid is achieved by a plant enzyme, oxalate oxidase. This enzyme has been characterized in some plant species including barley, beet and sorghum. The aim of our work is to isolate an oxalate oxidase gene and to introduce this gene in plant species susceptible to Sclerotinia sclerotiorum. We have purified to homogeneity oxalate oxidase from barley seedlings or from commercial preparations and antibodies were raised into rabbits. The protein was digested by CnBr and two internal peptides and the N-terminus were sequenced. These sequences share a very strong homology (97%) with a wheat protein of unknown function unduced during germination and called germin. To confirm the identity between oxalate oxidase and germin, the biochemical properties of the two proteins were compared. As germin, oxalate oxidase is glycosylated and is a tetramer of 100 kD resistant to SDS denaturation.