Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics.
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.
Grimaldi
Benedetto
B
Dipartimento di Genetica e Biologia Molecolare, Universitá di Roma "La Sapienza", P. le Aldo Moro 5, Roma 00185, Italy.
de Raaf
Michiel A
MA
Filetici
Patrizia
P
Ottonello
Simone
S
Ballario
Paola
P
D016960:Agrobacterium tumefaciens / Q000187:drug effects / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001203:Ascomycota / Q000235:genetics*; D002934:Cinnamates / Q000494:pharmacology; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004351:Drug Resistance / Q000235:genetics; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005822:Genetic Vectors; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D004271:DNA, Fungal; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A