The Transformation and Protein Expression of the Edible Mushroom Stropharia rugosoannulata Protoplasts by Agrobacterium-tumefaciens-Mediated Transformation

<i>Stropharia rugosoannulata</i> is a cultivated edible mushroom characterized by its nutritional composition and efficient cellulolytic enzymatic systems. However, the lack of genetic tools has significantly impeded the investigation of its molecular mechanisms, severely constraining the study of functional genomic and precision breeding in <i>S. rugosoannulata</i>. It was demonstrated in this study that the <i>Agrobacterium-tumefaciens</i>-mediated genetic transformation (ATMT) system is applicable for the transformation of <i>S. rugosoannulata</i> protoplasts. Through this proposal, we successfully achieved the expression of exogenous genes (<i>mCherry</i> gene encoding red fluorescent protein, <i>hph</i> gene encoding hygromycin B phosphotransferase, and <i>GUS</i> gene encoding β-glucuronidase) and the endogenous mutant gene <i>SDI</i> encoding the iron-sulfur protein subunit of succinate dehydrogenase in <i>S. rugosoannulata</i>. Furthermore, this study employed endogenous promoters of <i>GPD</i> encoding glyceraldehyde-3-phosphate dehydrogenase and <i>SDI</i> to enhance transformation efficiency and drive target gene expression. This study establishes the feasibility of ATMT in <i>S. rugosoannulata</i> systems, while achieving stable expression of a panel of selectable marker genes and reporter genes critical for genetic research in <i>S. rugosoannulata</i>.