We have used a plasmid containing the argB gene to transform an Aspergillus nidulans argB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ~ 3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.
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Acknowledgements This work was partially supported by grants 0708-N9109 from CONACyT, and IN200192 from DGAPA-UNAM, Mexico. We thank Gabriela Soid for the diploid complementation tests. We also thank Thomas Adams and Wilhelm Hansberg for critical reading of the manuscript.
- Asexual development
- Gene tagging
- Restriction enzyme-mediated integration