Functional characterization of the N assimilation pathways in the mycelium of Laccaria bicolor and the ectomycorrhizal symbiosis

Abstract Ectomycorrhizal (ECM) fungi contribute to N, Pi, and water uptake in trees while obtaining carbohydrates from their host plants. However, the molecular mechanisms underlying N assimilation during ECM symbiosis remain unclear. In this study, we used RNA interference (RNAi) to silence the expression of genes encoding glutamine synthetase (GS) and NADP-glutamate dehydrogenase (GDH), which are key enzymes involved in N assimilation in the ectomycorrhizal basidiomycete, Laccaria bicolor. LbGS and LbGDH RNAi strains exhibited significantly reduced mycelial growth when cultivated with various inorganic N sources. Compared with the wild-type mycelium, the RNAi strains demonstrated a reduced formation rate of ECM rootlets, indicating the essential role of these 2 enzymes in the establishment of symbiosis. Transcriptomic analysis revealed that silencing of LbGS and LbGDH also altered the expression of other genes involved in N metabolism in ECM rootlets. 15N and 13C tracer experiments demonstrated that LbGS silencing affects carbon exchange in ECM roots. Our findings have established that both GS and NADP-GDH pathways play crucial roles in N assimilation in free-living mycelia and ECM roots, although the GS/GOGAT pathway appears to be predominant.