Bioluminescence in the Edible Mushroom Hypsizygus marmoreus by Transformation with a Fungal Luciferase Gene

Following the elucidation of the fungal bioluminescence pathway (FBP), it was quickly adopted as a reporter system in plants; however, no such application has been documented in fungi to date. In this study, we established for the first time a luminescent reporter in the commercially important mushroom Hypsizygus marmoreus by expressing the luciferase gene from the luminous fungus Neonothopanus nambi. Using an established Agrobacterium-mediated transformation method, we separately introduced the wild-type luciferase gene nnLuz and the previously reported optimized variant nnLuz-v4 that can enhance bioluminescence expression into H. marmoreus arthroconidia. Both genes were stably integrated into the genome and expressed under the control of the H. marmoreus Glycerol 3-phosphate dehydrogenase (GPD) gene promoter. Upon addition of exogenous luciferin, transformants carrying the wild-type nnLuz produced clear, readily detectable bioluminescence signals, whereas no luminescence was observed in untransformed controls. Unexpectedly, the wild-type luciferase consistently exhibited substantially higher luminescence intensity than the optimized nnLuz-v4 variant. This finding suggests that codon optimization may be unnecessary or even detrimental when the donor and host are phylogenetically close basidiomycetes. The successful deployment of the fungal luciferase gene in H. marmoreus provides a sensitive and non-invasive genetic tool that does not require external excitation. This system opens new avenues for promoter characterization, real-time gene expression monitoring during mushroom development, and molecular breeding efforts aimed at improving agronomically important traits.