Enhanced Transformation inMagnaporthe griseaby Restriction Enzyme Mediated Integration of Plasmid DNA

Abstract

To use insertional mutagenesis for gene tagging, we tested whether transformation of Magnaporthe grisea could be enhanced by restriction enzyme mediated integration (REMI) of plasmid DNA. The integrative plasmid pAN7-2 was linearized by restriction endonucleases HindIII, BamHI, BglII, and EcoRV, and used to transform M. grisea strain Guy11 in the presence of the corresponding enzyme. Transformation frequency was increased by the incorporation of restriction enzymes, but the level of enhancement appeared to be enzyme-specific. An increase of up to 10-fold (75-200 transformants/μg DNA) was achieved with BamHI and BglII, but only a two-fold increase was observed with HindIII. The optimal enzyme concentration for enhanced transformation also varied among enzymes (e.g., 20 units per milliliter for BamHI vs. 200 units per milliliter for HindIII). Transformation efficiency appeared to be correlated with frequency of REMI. Southern blot analysis of the BamHI-transformants showed that 72% of the plasmid integrations were at BamHI sites whereas 28% of the integrations in HindIII-transformants were at HindIII sites. In EcoRV-mediated transformation, 42% of the integrations were at EcoRV sites. REMI transformation has allowed us to generate a large number of random insertions in the fungal genome from which mutant genes can be isolated