An hydroxynaphtalene reductase gene from the wood-staining fungus Ophiostoma floccasum complements the buff phenotype in Magnaporthe grisea

To understand the mechanism of wood discolouration employed by the staining fungus Ophiostoma floccosum, a gene involved in dihydroxynaphthalene (DHN) melanin biosynthesis was cloned from this organism and its function was examined. Degenerate oligonucleotide primers were designed according to conserved regions of hydroxynaphthalene (HN) reductases and used to PCR-amplify a 380 bp fragment from genomic DNA of O. floccosum. The amplified product was used as a probe to screen an O. floccosum genomic phage library. A genomic clone containing a full-length HN reductase gene was retrieved and a total of 1681 bp of its nucleotide sequence was determined. The determined sequence contained 673 bp of 5' untranslated sequence, 807 bp of putative open reading frame (ORF) encoding a protein of 269 amino acids and 197 bp 3' untranslated sequence. The ORF contained a 76-bp intron. Based on the similarity of the inferred amino acid sequence to other known fungal HN reductases, the cloned O. flocosum gene was considered an HN reductase gene. Disruption of the O. floccosum reductase gene was unsuccessful due to integration anomalies during homologous recombination. As an alternative, we complemented an HN reductase deficient buf mutant of the rice blast fungus Magnaporthe grisea by introducing the cloned O. floccosum reductase gene using hygromycin B resistance plasmid pCB1004. The complemented M. grisea buf mutants produced a black pigment like a wild type strain, indicating that the O. floccosum reductase is functionally homologous to other fungal HN reductases. These results suggest that O. floccosum uses the DHN pathway for pigmentation.