A second reductase gene involved in melanin biosynthesis in the sap-staining fungus Ophiostoma floccosum

Abstract.

Two different DNA sequences amplified by PCR from Ophiostoma floccosum show high homology to reductase genes of the fungal melanin biosynthetic pathway and suggest that two reductases may participate in this pathway in O. floccosum. Previously, one reductase gene, THN1 had been isolated from O. floccosum and proved to be involved in melanin biosynthesis. The second reductase gene, THN2, encodes a protein of 284 amino acids and is the longest fungal melanin reductase so far reported. The protein sequences deduced from the two THN genes show 44% identity. The products of THN1 and THN2 belong to two different groups of melanin reductases based on a comparative analysis of sequences from several fungal species. We confirmed the function of the THN2 gene by complementing dihydoxynaphthalene (DHN)-melanin-deficient, non-pathogenic mutants of Colletotrichum lagenarium and Magnaporthe grisea that lack the 1,3,8-trihydroxynaphthalene reductase. THN2 transcripts were detected in these transformants. These melanin-producing transformants showed mycelial and appressorium melanization. C. lagenarium transformants that formed melanin exhibited pathogenicity. This suggests that the cloned THN2 gene product functions as the 1,3,8-trihydroxynaphthalene reductase in the DHN melanin biosynthetic pathway in both C. lagenarium and M. grisea.