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Spatio-temporal regulation of pre-symbiotic signalling in arbuscular mycorrhizal symbiosis https://doi.org/10.17863/CAM.124963 Ferreras Garrucho, Gabriel 0000000245250439 Paszkowski, Uta Arbuscular mycorrhizal (AM) symbiosis is a ubiquitous and ancient interaction between plant roots and Glomeromycotina fungi, underpinned by a mutualistic nutrient exchange. In this intimate partnership, the fungus dynamically and asynchronously colonises the plant root system and cortex cells. Symbiosis development must therefore require spatio-temporally fine-tuned molecular control mechanisms. Although the plant genetic programme underlying AM symbiosis has been extensively studied, little is known about its dynamic regulation across root cell layers and developmental stages of the association. Early signalling events are particularly understudied. This PhD work therefore aimed to investigate the spatio- temporal dynamics of AM pre-symbiotic signalling. A combination of genetic, systems biology and microscopic technologies were leveraged for this purpose. Plant symbiotic competence is under the control of the DWARF14-LIKE (D14L) signalling pathway, yet when and where the genetic components of this pathway are functionally required is largely unknown. Activity of the D14L promoter, well as those of downstream partners D3 and SMAX1, was found to be surprisingly variable between roots and cell-types, primarily but not strictly active in the root vasculature. However, cell-type specific complementation revealed that D14L is mostly required in outer cell layers for its role in AMS. Subcellularly, D14L was found to be nucleo-cytoplasmic, while downstream partners are exclusively nuclear. Surprisingly, both pools appeared to be separately functional in AMS. Results thus demonstrate that the spatial distribution of D14L is an important determinant of AM symbiosis. The signalling events behind perception of AM fungi are also unresolved, particularly whether chemical dialogue is sufficient for specific recognition or further layers of regulation are required. This was addressed by investigating the transcriptome of rice roots exposed to the complete suite of secreted signals from beneficial and pathogenic fungi. A diagnostic symbiotic response was only elicited under physical contact with AMF. Short or long-term exposure to beneficial or pathogenic fungal exudates conversely resulted in a non-specific general stress response partially overlapping with pattern-triggered immunity, independent of AM signalling components. Data thus revealed that rather than specific recognition in the rhizosphere, a sequence of signals orchestrates stress, immunity and symbiosis, pivoting towards symbiosis potentially at the stage of plant-fungal contact formation. Even during intraradical accommodation, despite extensive knowledge on the transcriptomic changes associated with fungal accommodation, many questions remain regarding stage and cell-type specific signatures. Translating Ribosome Affinity Purification (TRAP) using stage-specific AM-inducible promoters allowed to isolate RNA populations from distinct cell- states through AM development. The resulting dataset highlighted significant spatiotemporal translational repatterning across AM development. This included repression, missed by bulk approaches, of nutrient transporters, defence markers and cell-wall biogenesis in the early and mature arbuscule, that dampens during arbuscule collapse. Overall, findings from this dissertation shed light on the spatiotemporal complexities of early AM signalling, advancing our understanding of the fine-scale molecular coordination of AM symbiosis, and opening new avenues for functional exploration. University of Cambridge 2025-09-22 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/395480 https://www.repository.cam.ac.uk/bitstreams/a0592fdf-7d0d-4652-8bf6-cfd3ce616c4d/download 3365562cbdc157ebab282bf12b69a427 https://www.repository.cam.ac.uk/bitstreams/fc75aec1-7768-447c-a59a-1cd9ee7598d4/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Plant-microbe interactions Transcriptomics Arbuscular mycorrhizal symbiosis Single-cell omics Spatio-temporal resolution Confocal microscopy Plant molecular genetics