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 (trp3iar) 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 a's such has potential for the transformation of other homobasidiomycetes, including the major edible species. In this study the trp3iar 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 trp3iar 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 preincubating in liquid media before selection, enabled the recovery of between 0.3 and 12 trp3iar transformants mug-1 of DNA. Although the background growth of non-transformed C. bilanatus proved difficult to eliminate, the expression of 5FI resistance in trp3iar 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 trp3iar. Novel dikaryons constructed between transformants and wild-type monokaryons were used to demonstrate that trp3iar behaved as a dominant gene in C. bilanatus.