Functional characterization of the T. melanosporum pheromone-receptor system by yeast complementation assays

The release of T. melanosporum genome has recently allowed us to identify the structure of the mating type locus (MAT) and in turn to find out that the two MAT genes at this locus are present in different strains (Martin et al., Nature 2010; Rubini et al., New Phytol 2011). This organization of the MAT locus typifies heterothallic ascomycetes. In these fungi mating occurs only between strains of opposite mating type, which attract each other by secreting pheromones. Each mating type produces its own specific pheromone These can be divided into two groups, ?- and a-factor pheromones. Each pheromone interacts with its cognate receptor synthesized by a strain of opposite mating type: the ?- and a-factor pheromones are bound by STE2 and STE3 receptors, respectively. This binding triggers the activation of a mitogen-activated protein kinase (MAPK) signaling pathway ultimately targeting a homeodomain transcription factor (STE12) that coordinates the mating program of the cell (Debuchy et al., ASM press 2010). Most of the key genes of the pheromone pathway have been in silico identified in the T. melanosporum genome, among them, the putative genes for the ??-factor (ppg1) and a-factor (ppg2) pheromones, similar to those of Saccharomyces cerevisiae, along with genes coding for their receptors (Martin et al., Nature 2010). Besides in silico identification, however, no functional evidences for the T. melanosporum pheromone receptor system have been provided yet. To achieve this goal, here we employed the yeast S. cerevisiae as a heterologous model system. Thus, the T. melanosporum coding sequences for the pheromone receptors STE2 and STE3 and for the putative pheromone precursors ppg1 and ppg2 have been isolated by RT-PCR from in vitro grown mycelia of opposite mating type and each one cloned in a yeast expression vector (pPGK, Kang et al., Cell Biol 1990). The pPGK vectors harboring the above mentioned truffle cDNAs were then transformed into suitable yeast strains lacking the endogenous pheromone and receptor genes. Agar diffusion bioassay (Halo assay) was used to evaluate the interaction between yeast strains carrying the heterologous pheromone and cognate receptor genes. Further to this, a synthetic ?-factor peptide, designed on the basis of the in silico analysis of the putative gene for ??pheromone precursor, was tested for its capacity in triggering morphological changes in a yeast strain expressing the respective truffle receptor gene (STE2). These experiments documented a positive interaction between the protein pair coded by the T. melanosporum ppg1 and STE2 genes. Thus, a functional evidence for a truffle pheromone-receptor system has been provided for the first time. Efforts are in progress to provide functional evidence for the a-factor pheromone and STE3 receptor system. The identification of pheromone and receptor genes in Tuber spp. is fundamental to deep our insight into the genetic mechanisms and environmental stimuli that control the fertilization process, and thus the fructification, of these fungi.