clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum

Abstract.

A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant. This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris. In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black. Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized. Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae. Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation. Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants. Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity. These observations suggest that the clap1 gene product may be involved in the pathogenicity of C. lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin.