Cloning and Characterization of a Melanin Biosynthetic THR1 Reductase Gene Essential for Appressorial Penetration of Colletotrichum Lagenarium

Melanin biosynthesis of Colletotrichum 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-trihydroxynaphthalene to vermelone in the melanin biosynthetic pathway. The mutant formed nonmelanized appressoria and had little infectivity on cucumber leaves. A cosmid clone pCR1 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. pCR1 transformed the Thr- mutant 9141 to wild-type phenotype. A DNA fragment (THR1) homologous to BRM2 was subcloned from pCR1 and the nucleotide sequence determined. THR1 contains one open reading 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 THR1 amino acid sequence contains a region highly similar to the Ver1 gene involved in the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis by Aspergillus parasiticus and to the T4HN reductase gene involved in the conversion of 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone in melanin biosynthesis by Magnaporthe grisea. Expression of the THR1 gene during spore germination of C. lagenarium was detected by RNA blotting. We propose that the C. lagenarium THR1 gene encodes a reductase involved in conversion of 1,3,8-trihydroxynaphthalene to vermelone.