Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in thermophilic fungus Thermomyces lanuginosus.

Objective: To establish a stable transformation system of the thermophilic fungus Thermomyces lanuginosus for its insertional mutagenesis.

Methods: Agrobacterium tumefaciens-mediated transformation (ATMT) was applied to establish transformation system of T. lanuginosus. Southern blotting of hph gene and cloning of transforming DNA (T-DNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome, respectively.

Results: A reliable transformation method is established for T. lanuginosus. Specifically, pre-germinating spores of T. lanuginosus used at co-cultivated period was a prerequisite. T. lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28 degrees C for 48 h achieved the highest transformation efficiency. Addition of Acetosyringone (AS) during pre-culture of A. tumefaciens and co-cultivation of T. lanuginosus germinating spores with A. tumefaciens was essentially required, and the best results were obtained with AS at the concentration of 500 microM. Southern blotting analysis showed that majority of transformants (79.2%) contained a single insertion of T-DNA. Thermal asymmetric interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal genome. Using the transformation system, some stable phenotypic mutants of T. lanuginosus were obtained.

Conclusion: We report, for the first time, a simple and efficient method for transforming T. lanuginosus by using ATMT. This approach provides a tool for insertional mutagenesis gene tagging in this thermophilic fungus.