Oxalate decarboxylase from Collybia velutips: molecular cloning and its over expression to confer resistance to fungal infection in transgenic tobacco and tomato

Abstract

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mecha- nism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5؅-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548 –23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants ex- pressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotio- rum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.