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The cloning and characterization of a THN reductase gene from ophiostoma floccosum 387n Eagen, Rebecca


Sapstaining fungi, in particular Ophiostoma sp., cause significant economic problems for Canada's lumber industry and current control methods are less than ideal. The visible stain found on lumber is caused by melanin produced in the mycelia of sapstaining fungi as they grow through the sapwood. O. floccosom 387N is frequently isolated from stained lumber but knowledge of this fungus is lacking. A molecular biological approach, in addition to elucidating important data about this organism, may provide information that could prove valuable in designing specific melanin inhibitors or creating pigment deficient strains that could be used for biocontrol. The novel application and adaptations of molecular biological techniques to O. floccosom 387N were used to isolate and characterize a gene from the melanin biosynthetic pathway. Culture conditions were defined during which pigmentation and biomass yields were significant. Application of dihydroxynaphthalene (DHN) pathway inhibitors caused the reduction of visible pigment production in 387N and allowed us to speculate that the DHN pathway of melanin biosynthesis was important in 387N. Hybridization data indicated that 387N possessed sequences that were very similar to other filamentous fungal DHN genes. A genomic library of 387N was screened and we recovered a gene with an 877 nucleotide open reading frame that coded for a protein of 268 amino acids. The protein shared similarity with other fungal tri- or tetra-hydroxynaphthalene (THN) reductases and shared determinative characteristics of the family of short chain alcohol dehydrogenases. To prove the function of the gene that was isolated and characterized, the 387N THN reductase gene was used to complement THN reductase deficient buf mutants of Magnaporthe grisea. Several transformants were recovered that had wild type pigmentation. Hybridization studies and reverse transcriptase - polymerase chain reactions (RT-PCR) indicated that these transformants had integrated copies of the 387N gene and that gene transcripts were present. This evidence led us to conclude that the gene isolated from 387N functions as a THN reductase.

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