Spatiotemporal regulation of arbuscular mycorrhizal symbiosis at cellular resolution.

Arbuscular mycorrhizal (AM) symbiosis develops through fungal colonization of root epidermal and cortical cells, culminating in the formation of arbuscules, transient, tree-like intracellular hyphal structures for nutrient exchange. To dissect the complexity of AM establishment in rice (Oryza sativa) roots colonized by Rhizophagus irregularis, we conducted spatial transcriptomics of plant and fungal genes at single-cell resolution. This revealed differences in transcriptional activity between fungal structures and reprogramming of plant cell-identity markers upon colonization. Furthermore, cells hosting similarly developed arbuscules showed striking transcriptional heterogeneity, suggesting hidden functional diversity at the individual cell level. For stage-resolved profiling of translation, we used AM-stage specific Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq) with promoters active at discrete stages of symbiosis or arbuscule development. This revealed extensive spatiotemporal changes in the ribosome-bound transcript population, including sets of phosphate, nitrogen, and carbon transporters and regulators with specific enrichment and depletion patterns at different stages of arbuscule development. Rice transcripts encoding cell wall biosynthesis genes and defense markers were present in low abundance at early stages but highly abundant at late stages of the arbuscule lifespan, supporting a host-driven shift toward arbuscule termination. Together, these findings highlight the nuanced 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.