Identification and Functional Analysis of Pheromone and Receptor Genes in the B3 Mating Locus of Pleurotus eryngii

Abstract

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

Current Technologies and Related Issues for Mushroom Transformation

“…Using this property, ATMT has been applied to the transformation of a variety of filamentous fungi, including Aspergillus, Fusarium, and Trichoderma [ 60 - 63 ]. Spores, mycelia, protoplasts, and tissues of fruiting bodies from basidiomycete fungi also have been subjected to ATMT ( Table 3 ) [ 61 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ]. Successful ATMT relies on several factors.…”

“…The concentration of acetosyringone, a wound response molecule required for activation of the vir gene [ 83 ], is normally maintained at 0.2 mM for most of the mushroom transformation ( Table 3 ). Duration and temperature for co-culture are normally 2~5 days and near 25℃, respectively, but can last up to a month at low temperature for slow-growing mushrooms [ 74 , 75 , 78 , 80 ]. Various binary vectors containing T-DNA regions, which are composed of insert DNAs planked by a left board and right board, have been applied to mushroom transformation.…”

Development of cultivation media for extending the shelf-life and improving yield of king oyster mushrooms (Pleurotus eryngii)

“…The cooled substrate was manually inoculated with 5 ± 0.1 g of spawn and incubated for 35 days in a dark incubation room maintained at 22 ± 1 • C and 65% relative humidity. The fruiting body was cultivated and harvested according to the method described by Kim et al (2014), with minor modifications. On the 35th day of incubation after inoculation, when mycelia were colonized most of the substrate in the bottles, the top media (<0.5 cm) and the old spawn were removed using a scraper (Segae Precision Co., Korea).…”

Mating-type genes and hyphal fusions in filamentous basidiomycetes

“…The knowledge of the B locus structure in C. cinerea and S. commune has facilitated its identification in tetrapolar basidiomycetes Laccaria bicolor (Niculita-Hirzel et al, 2008), Flammulina velutipes (van Peer et al, 2011, Lentinula edodes (Wu et al, 2013) and Pleurotus eryngii (Kim et al, 2014). In all these fungi, receptor and pheromone genes were identified in the B mating type locus.…”

“…Agrobacteriummediated transformation was recently applied for screening the B mating type genes in P. eryngii (Kim et al, 2014). Furthermore, the detailed analysis of well-known C. cinerea and S. commune mutant strains with self-activated B pathway demonstrated that mutations in receptor encoding genes in C. cinerea (Olesnicky et al, 1999(Olesnicky et al, , 2000 and in S. commune pheromone encoding genes (Fowler et al, 2001) were the reason for the activation of the B pathway and the exceptional mating behavior.…”