Cell Death Regulation and Function inPlant-Fungal Symbiosis

Regulated cell death (RCD) is a hallmark of plant immunity, traditionally associated with defense against pathogens. However, its role in beneficial plant–microbe interactions remains largely uncharacterized. The root endophyte Serendipita indica, with its biphasic lifestyle, comprising an initial biotrophic phase followed by a cell death-associated phase, serves as an excellent model to dissect the molecular regulation of RCD in mutualistic contexts. While individual host factors implicated in the cell death-associated phase have been previously identified, the mechanisms governing RCD initiation and regulation remained poorly defined. In this thesis (Chapter 4), I characterized the synergistic activity of two fungal effector enzymes, SiNucA and SiE5NT, as the primary trigger of RCD. Their activity leads to the apoplastic production of deoxyadenosine (dAdo), a purine-derived infochemical that triggers signaling and RCD in host roots. I further identify the equilibrative nucleoside transporter ENT3 as the key mediator of both dAdo-induced and S. indica-mediated cell death. To dissect the molecular responses underlying this process, integrated transcriptomic, proteomic, and metabolomic profiling were employed. These analyses revealed substantial overlap with known RCD pathways and uncovered additional infochemical candidates involved in host–microbe communication. To monitor cell death dynamics in planta, I developed a high-throughput assay combining Pulse Amplitude Modulation (PAM) fluorometry with ion leakage measurements (Chapter 3). A genetic screen using this assay identified a previously uncharacterized TIR-NLR receptor Induced by S. indica (ISI) as a modulator of root cell death and a regulator of dAdo-triggered RCD, supporting the role of dAdo as an active immunometabolite. Beyond this, ISI was found to modulate distinct root cell death phenotypes and to influence S. indica colonization patterns, suggesting a broader role in regulating mutualistic interactions. Together, these findings support a model in which synergistic effector activity and purine-based signaling tightly regulate RCD as part of a controlled mutualistic program. This work redefines RCD as a functional component of beneficial symbioses used to shape microbial niche structure, uncovers a novel intersection between purine metabolism and immune signaling, and broadens the functional repertoire of TIR-NLR proteins beyond classical pathogen resistance (Chapter 5). These findings advance the emerging field of plant immunometabolism and provide a conceptual framework for understanding how root immunity integrates metabolic and microbial cues.