title: Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.
abstract: The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.
authors.affiliation: Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, 5000, Summerland, BC, Canada, V0H 1Z0.
authors.forename: Shawkat
authors.initials: S
authors.lastname: Ali
authors.forename: Guus
authors.initials: G
authors.lastname: Bakkeren
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D016681:Genome, Fungal; D059765:Homologous Recombination; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D019076:Transgenes; D014588:Ustilago / Q000235:genetics*
chemical_list: D004271:DNA, Fungal