Nucleus-directed fluorescent reporter system for promoter studies in the ectomycorrhizal fungus Laccaria bicolor

Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.