Abstract
Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10(-4) was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25-30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.