Development of an Agrobacterium tumefaciens-mediated transformation system for Tilletia controversa Kuhn

Dwarf bunt of wheat caused by Tilletia controversa Kuhn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 mu mol/l, hygromycin B at 25 mu g/ml, 1 x 10(6) T. controversa hypha cells/ml, A. tumefaciens with OD600 of 0.5 co-cultivation at 16 degrees C for 48 h and culture was incubated at 16 degrees C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.