Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts.

The aim of the study was to evaluate the possibility of supplementation with inorganic forms of selenium (Na2SeO4 and Na2SeO3) in concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of three medicinal mushroom species: Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum. Tested mushroom species grew in Se additions of 0-0.6 mM (A. aegerita and H. erinaceus), while growth of G. lucidum bodies was observed for 0-0.8 mM. For the latter mushroom species, the total Se content was the highest. Content of Seorg was diverse; for control bodies it was the highest for G. lucidum (only organic forms were present), lower for A. aegerita (84% organic forms) and the lowest for H. erinaceus (56% organic forms). Accumulation of Se(IV) was generally significantly higher than Se(VI) for all tested mushroom species. There was no significant decrease of A. aegerita or G. lucidum biomass with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 ± 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 ± 8.73 g), control (191.80 ± 6.06 g) and 0.1 mM (185.04 ± 8.73 g) of both inorganic salts. The addition to the medium of Se salts brought about macroscopic changes in the fruiting bodies of the examined mushrooms. Concentrations exceeding 0.4 mM caused diminution of carpophores or even their total absence. In addition, colour changes of fruiting bodies were also recorded. At Se concentrations of 0.4 and 0.6 mM, A. aegerita fruiting bodies were distinctly lighter and those of H. erinaceus changed colour from purely white to white-pink.