Development of a transformation system for the edible mushroom Grifola frondosa: Demonstrating heterologous gene expression and RNAi-mediated gene silencing

• Transformation vectors for Grifola frondosa were constructed.
• Using this vector system, we successfully expressed EGFP and Luc in G. frondosa.
• RNAi-mediated gene silencing in G. frondosa was achieved using this vector system.

Grifola frondosa is a well-known edible mushroom that is industrially produced year-round under artificial cultivation. To improve the productivity and quality of cultivated mushrooms, transcriptomic and genomic data for G. frondosa have been used to investigate genes involved in fruiting body differentiation. However, further functional analysis of genes has been limited by the lack of specific genetic engineering tools for this species. Hence, the primary goal of this study is to establish an efficient transformation method and vector system for G. frondosa. To express transgenes efficiently, a transformation vector was constructed using homologous regulatory sequences from constitutively expressed genes identified from comprehensive gene expression profile of G. frondosa. Using this vector system, we have successfully expressed enhanced green fluorescent protein (EGFP) and firefly luciferase (Luc) in G. frondosa. Furthermore, we have tested RNA interference (RNAi)-mediated gene silencing using an RNAi vector, and showed that expression of the hairpin RNA (hpRNA) encoded by this vector can trigger downregulation of expression of Gf.HydA1, a model target hydrophobin gene from G. frondosa. Thus, the transformation system established in this study should be a useful tool for further functional analysis of other genes in G. frondosa.