Transformation of the homobasidiomycete Coprinus bilanatus to 5-fluoroindole resistance using a mutant trp3 gene from Coprinus cinereus

A dominant mutation in the anthranilate synthetase gene ( trp3) of Coprinus cinereus confers resistance to the antimetabolite 5-fluoroindole (5FI). The mutant gene ( trp3 iar) can be used as a positively selectable marker for the transformation of C. cinereus. It is the first positively selectable marker gene of homobasidiomycete origin that has been cloned, and as such has potential for the transformation of other homobasidiomycetes, including the major edible species. In this study the trp3 iar gene was used to develop heterologous transformation of the model secondarily homothallic species, Coprinus bilanatus. Three different methods were tested for the selection of C. bilanatus trp3 iar transformants. Overpouring regenerating protoplasts with soft agar containing 5FI was not effective in inhibiting the growth of non-transformed cells. Two other methods, plating directly on to inhibitory media or pre-incubating in liquid media before selection, enabled the recovery of between 0·3 and 12 trp3 iar transformants g −1 of DNA. Although the background growth of non-transformed C. bilanatus proved difficult to eliminate, the expression of 5FI resistance in trp3 iar transformants could be used to identify the genuine transformants in a secondary screen. Southern analyses of several transformants showed a similar hybridization pattern, suggesting preferential sites for the integration of transforming trp3 iar. Novel dikaryons constructed between transformants and wild-type monokaryons were used to demonstrate that trp3 iar behaved as a dominant gene in C. bilanatus.