Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

Andrea Brenna,Simone Ottonello,Paola Ballario,Eleonora Muggiano,Barbara Montanini,Patrizia Filetici,Marco Proietto

doi:10.1186/s13568-014-0043-x

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.

Highlights

IntroductionThe ascomycete Tuber borchii is a hypogeous fungus (genus, Tuber; family Tuberaceae; order Pezizales) that establishes a beneficial mutualistic interaction (‘ectomycorrhiza’) with the roots of many tree species
The ascomycete Tuber borchii is a hypogeous fungus that establishes a beneficial mutualistic interaction (‘ectomycorrhiza’) with the roots of many tree species
We previously described an Agrobacterium tumefaciens-mediated (ATM) transformation procedure for T. borchii (Grimaldi et al 2005), which due to the insertion of transgenic DNA driven by the transfer DNA (T-DNA), and consequent lack of stability, could not be effectively exploited for functional genomics studies

Summary

Introduction

The ascomycete Tuber borchii is a hypogeous fungus (genus, Tuber; family Tuberaceae; order Pezizales) that establishes a beneficial mutualistic interaction (‘ectomycorrhiza’) with the roots of many tree species. T. borchii has a pronounced saprobiotic capacity and can be grown in vitro (albeit quite slowly) as vegetative free-living mycelium in the absence of a plant host. This mixed symbiotic/saprophytic lifestyle (Hebe et al 1999), together with the lack of asexual spores amenable to in vitro culture has hampered the genetic manipulation of this fungus and obscured our understanding of its complex life cycle. Tuber fruitbodies (‘truffles’) lack an active system for the dispersal of spores, which are disseminated by the action of mycophagous animals (Pegler et al.1993). T. borchii is the truffle that can be more handled under laboratory conditions

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