Cloning and Characterization of a Melanin Biosynthetic THR1 Reductase Gene Essential for Appressoria! Penetration of Colletotrichum lagenarium. N. S. Perpetua. Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan. Y. Kubo, N. Yasuda, Y. Takano, and I. Furusawa Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan. MPMI 9:323-329. Accepted 4 March 1996. Copyright 1996 The American Phytopathological Society.

Melanin biosynthesis of Collelotrichum lagenarium is essential for appressorial penetration of the host plant. A melanin deficient mutant 9141 (Thr^-) has a defect in the conversion of 1,3,8-trihydroxynaphthaIcne to vermelone in the melanin biosynthetic pathway. The mutant formed nonmelanized appressoria and had little infectivity on cucumber leaves. A cosmid clone pCRl was selected from a cosmid library of wild-type C. lagenarium by means of a heterologous probe BRM2, one of the clustered genes involved in melanin biosynthesis of Alternaria alternata. pCRl transformed the Thr^- mutant 9141 to wild-type phenotype. A DNA fragment (THRI) homologous to BRM2 was subcloned from pCRl and the nucleotide sequence determined. THRI contains one open rea'ding frame that encodes a protein of 282 amino acids. A transformant resulting from gene disruption showed a light brown phenotype different from the dark brown phenotype of the wild-type 104-T. The transformant formed nonmelanized appressoria and had little infectivity. The THRI amino acid sequence contains a region highly similar to the Verl gene involved in the conversion of versicolorin A to sterigmato-cystin in aflatoxin biosynthesis by Aspergillus parasilicus and to the T₄HN reductase gene involved in the conversion of 1,3,6,8-tetrahydroxynaphthaIene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone in melanin biosynthesis by Magnaporthe grisea. Expression of the THRI gene during spore germination of C. lagenarium was detected by RNA blotting. We propose that the C. lagenarium THRI gene encodes a reductase involved in conversion of 1,3,8-trihydroxynaphthalene to vermelone