Martyn, A.; Thorsgaard Jensen, I.; Lind Salomonsen, C.; Blahovska, Z.; Tao, K.; Dings, H.; Nott Bjoergvinsdottir, T.; Tolstrup Christensen, B.; Oldroyd, G.; Waagepetersen, R.; Glasius, M.; Radutoiu, S.

Crop nutrition depends on plant-microbe interactions, yet it remains unclear whether conserved genetic pathways impose universal rules on root microbiome assembly across plant hosts. Here, we show that the Common Symbiosis Signalling Pathway (CSSP), a conserved genetic module controlling endosymbiosis with arbuscular mycorrhizal fungi and nitrogen-fixing bacteria, regulates root microbiome assembly in a host-specific manner across contrasting fertilisation regimes. Using Lotus japonicus and Hordeum vulgare, we demonstrate that mutations in orthologous CSSP genes remodel root bacterial communities in both species, but with distinct taxonomic outcomes. In Lotus, CSSP disruption reduces rhizobial colonisation and promotes niche replacement by commensal taxa, whereas in Hordeum, the same mutations broadly restructure bacterial lineages without converging on Lotus-like responses. Root exudate profiling reveals host-specific metabolic differences, particularly in phenylpropanoid (flavonoids and coumarins) and gibberellin pathways, linking CSSP activity to chemically distinct rhizosphere environments that correlate with divergent microbiome assembly patterns across hosts. Moreover, root bacterial community composition accurately predicts plant nutritional status, highlighting tight coupling between host physiology and microbiome composition. Together, our results show that conserved symbiosis signalling regulates root microbiome assembly, while host-specific metabolic environments determine taxonomic outcomes. This extends CSSP function beyond canonical endosymbioses and positions symbiosis signalling as a general determinant of plant-microbiome interactions with implications for crop nutrition.