Agrobacterium tumefaciens-mediated genetic transformation of the ect-endomycorrhizal fungus terfezia boudieri

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD₆₀₀; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.