Functional characterization of the N assimilation pathways in the mycelium of <i>Laccaria bicolor</i> and the ectomycorrhizal symbiosis

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.