Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei

Penicillium marneffei is an opportunistic fungal pathogen of humans, causing respiratory, skin, and systemic mycosis in south-east Asia. Here we describe the transformation of P. marneffei with Agrobacterium tumefaciens, and the optimization of the transformation procedure. Transformations in different combinations between A. tumefaciens stains (LBA4404 and EHA105) and binary vectors (pCB309A, pBI129A, and pCaMBIA1312A) showed that EHA105/pBI129A were the most efficient partners. Southern blot analysis suggested that 87.5 % of transformants obtained with this protocol displayed single hybridization bands, indicating a single insert of T-DNA in each of the transformants. Unique hybridization patterns, along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in P. marneffei. Several mutants with altered phenotypes were obtained during the construction of the mutant library, indicating the usefulness of the approach for functional genetic analysis in this important fungal pathogen.