Spatiotemporal regulation of arbuscular mycorrhizal symbiosis at cellular resolution

Arbuscular mycorrhizal (AM) symbiosis develops through successive colonization of root epidermal and cortical cells, culminating in the formation of arbuscules, tree-like intracellular structures that are transient yet essential sites of nutrient exchange. To dissect the cellular and structural complexity of AM establishment in rice roots colonized by Rhizophagus irregularis, we applied dual-species spatial transcriptomics to simultaneously monitor plant and fungal gene transcripts at single-cell resolution. This approach revealed surprising differences in transcriptional activity between fungal structures and showed that morphologically similar arbuscules can be transcriptionally distinct. These findings suggest hidden functional diversity among arbuscules at single-cell resolution. Because arbuscules form and degenerate within only a few days, we further sought to capture translational activities across their life span. We pioneered AM-inducible TRAP-seq (Translating Ribosome Affinity Purification followed by RNA-seq) using stage-specific promoters, enabling cell-type- and stage-resolved profiling in AM symbiosis. This revealed extensive spatiotemporal reprogramming of nutrient transport and signalling, with distinct sets of phosphate, nitrogen, and carbon transporters and regulators induced or repressed at different stages of arbuscule development, suggesting that nutrient exchange is dynamically regulated across the arbuscule life cycle. More broadly, cell wall biosynthesis genes and key defence markers were suppressed during arbuscule formation, whereas at a later stage, defence markers were strongly upregulated, suggesting a host-driven shift towards arbuscule termination. Together, these findings highlight the nuanced and dynamic regulation of AM symbiosis at the cellular level, refining our understanding of how nutrient exchange and fungal development are coordinated in space and time.