title: Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.
abstract: Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.
authors.affiliation: Pasteur Cenci Bolognetti Foundation, c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Piazzale A. Moro 5, Rome, 00185, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brenna
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, Laboratory of Functional Genomics and Protein Engineering, University of Parma, Parma, 43124, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Montanini
authors.affiliation: Pasteur Cenci Bolognetti Foundation, c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Piazzale A. Moro 5, Rome, 00185, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Muggiano
authors.affiliation: Pasteur Cenci Bolognetti Foundation, c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Piazzale A. Moro 5, Rome, 00185, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Proietto
authors.affiliation: Institute of Biology and Molecular Pathology, CNR, Rome, 00185, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Filetici
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, Laboratory of Functional Genomics and Protein Engineering, University of Parma, Parma, 43124, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Ottonello
authors.affiliation: Pasteur Cenci Bolognetti Foundation, c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Piazzale A. Moro 5, Rome, 00185, Italy ; Institute of Biology and Molecular Pathology, CNR, Rome, 00185, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Ballario
keywords: Agrobacterium tumefaciens-mediated transformation; Green fluorescent protein; Hygromycin phosphotransferase; T-DNA; TAIL-PCR; Truffles; Tuber spp; binary plasmid