EFFICIENT GENE TARGETING IN THE FILAMENTOUS FUNGUS ALTERNARIA-ALTERNATA

To characterize homologous recombination of transforming DNA in the fi lamentous fungus Alternaria alternata, we have compared the frequencie s of gene targeting by circular and linear DNA fragments in the fungus . The A. alternata BRM1 gene, which is an essential gene for melanin b iosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycel ial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid conta ining the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1(-)) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy(R)) transformants, correlating cl osely with the size of the BRM1 segment in the transforming DNA. Restr iction enzyme digestion within the BRM1 region greatly enhanced the fr equency of gene targeting: integration of the linear plasmids was almo st completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments an d rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1(-) transformants accounted for only 5% of Hy(R ) transformants. In contrast, when the linear plasmid produced by rest riction enzyme digestion within the BRM1 segment was used, almost all transformants were Me1(-). These results indicate that homologous inte gration of circular molecules in A. alternata is sensitive to the leng th of homology and the number of targets, and that double-strand break s in transforming DNA. greatly enhance homologous recombination.