Functional specialisation of ammonium transporters in the ectomycorrhizal fungus <i>Laccaria bicolor</i>

Ectomycorrhizal (ECM) fungi enhance nitrogen (N) uptake in trees; however, the molecular mechanisms and functional specialisation among transporter isoforms remain poorly understood. Here, we characterised two ammonium transporters, LbAMT1.1a and LbAMT2.3, in the basidiomycete Laccaria bicolor, revealing complementary roles in fungal growth and symbiosis. Transcriptomic analysis revealed that LbAMT1.1a was constitutively expressed during mycelial growth, whereas LbAMT2.3 was specifically induced during ECM formation. RNAi targeting each gene reduced mycelial growth, with LbAMT1.1a silencing producing stronger defects. ¹⁵N-ammonium tracing demonstrated that both RNAi strains exhibited an over 83% reduction in ammonium uptake compared to wild-type. In planta experiments revealed differential impacts on symbiosis. Both RNAi strains showed a 31-60% reduction in ECM formation. LbAMT2.3 RNAi significantly reduced lateral root formation, suggesting an additional role in developmental signalling. Gene expression analysis revealed that LbAMT2.3 silencing suppressed LbAMT1.1a transcript levels, indicating regulatory crosstalk between subfamilies. Dual isotope tracing (¹⁵N/¹³C) confirmed that impaired fungal N uptake reduces both N transfer and carbon allocation, with LbAMT1.1a disruption having a greater impact. In conclusion, LbAMT1.1a serves as the primary ammonium uptake pathway, whereas LbAMT2.3 functions as both a symbiosis-induced transporter and a positive regulator of LbAMT1 family expression, with an additional role in modulating host root architecture.