CLONING OF A MELANIN BIOSYNTHETIC GENE ESSENTIAL FOR APPRESSORIAL PENETRATION OF COLLETOTRICHUM-LAGENARIUM

We constructed a cosmid vector pKV-beta-for isolating genes by complementation of mutations in Colletotrichum lagenarium. pKV-beta-contains the bacteriophage lambda-cos site and the benomyl-resistant C. lagenarium beta-tubulin gene as a selective marker for Colletotrichum transformation. A genomic DNA library of wild-type C. lagenarium was constructed in pKV-beta. An albino mutant strain 79215 was transformed with DNA from this library and benomyl-resistant transformants were obtained at frequencies of approximately 20 transformants per microgram of DNA. Seven melanin-restored transformants were obtained from approximately 10,000 benomyl-resistant transformants. Albino mutants of C. lagenarium form nonmelanized appressoria and possess little penetrating ability. However, the transformants formed melanized appressoria with the ability to penetrate as efficiently as in the wild-type strain. From genomic DNA of a melanin-restored transformant integrated cosmid sequences (pAC7) were recovered by transduction of Escherichia coli to ampicillin resistance following treatment in vitro with lambda packaging extract. pAC7 transformed albino mutant 79215 to a melanin-restored wild phenotype with high frequency. From structural analysis of pAC7, an 8.4-kb BamHI fragment of pAC7 contains a wild-type copy of the gene involved in albino phenotype.