title: 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 1x10(4) Hygromycin B resistant colony forming units (CFU) have been achieved on each 90mm agar plate using 10(6) 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.
authors.affiliation: Biomaterials and Biocatalysts Group, Temasek Life sciences Laboratory, Research Link, National University of Singapore, Singapore 117604, Singapore. jilh@tll.org.sg <jilh@tll.org.sg>
authors.forename: Lianghui
authors.initials: L
authors.lastname: Ji
authors.forename: Zi-De
authors.initials: ZD
authors.lastname: Jiang
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Liu
authors.forename: Chong Mei John
authors.initials: CM
authors.lastname: Koh
authors.forename: Lian-Hui
authors.initials: LH
authors.lastname: Zhang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D005615:Freezing; D005800:Genes, Fungal; D005822:Genetic Vectors; D006921:Hygromycin B / Q000494:pharmacology; D008967:Molecular Biology / Q000379:methods*; D009172:Mycology / Q000379:methods*; D012641:Selection, Genetic; D014170:Transformation, Genetic; D014588:Ustilago / Q000235:genetics*
chemical_list: D000935:Antifungal Agents; D004269:DNA, Bacterial; C036483:T-DNA; D006921:Hygromycin B