A Simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells

Abstract: Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1×104 Hygromycin B resistant colony forming units (CFU) have been achieved on each 90mm agar plate using 106 sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5kb from the coding sequences at the 5′ UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis. [Copyright &y& Elsevier]