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Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics

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Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as “truffles”. A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii.

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We thank Peter Romaine (Department of Plant Pathology, The Pennsylvania State University) for the gift of the AGL-1 A. tumefaciens strain and the binary plasmid pBGgHg, Enzo Marchetti (Dipartimento di Genetica e Biologia Molecolare, Università di Roma “La Sapienza”) for assistance with confocal microscopy and Alessandra Gatti (Dipartimento di Genetica e Biologia Molecolare, Università di Roma “La Sapienza”) and Elisabetta Soragni (Center for Molecular Genetics, University of California, San Diego) for their help in initial phases of this work. Partial support from the Institute Pasteur, Fondazione Cenci Bolognetti and from the CNR Project “Tuber: biotecnologia dei funghi eduli ectomicorrizici” and FIRB RBNE011KMT9 (P.B.) and from the Regione Emilia-Romagna and the FIRB program “Genomica funzionale dell’interazione tra piante e microrganismi“ (S.O.) is also gratefully acknowledged.

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Correspondence to Paola Ballario.

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Communicated by U. Kück

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Grimaldi, B., de Raaf, M.A., Filetici, P. et al. Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics. Curr Genet 48, 69–74 (2005).

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