P-387 - Spatiotemporal dynamics of calcium signaling during resistosome-mediated cell death

Activation of many plant nucleotide-binding domain leucine-rich repeat (NLR) proteins triggers the formation of resistosomes, an NLR complex that mediates calcium influx and ultimately induces hypersensitive cell death. However, the precise spatiotemporal dynamics of calcium signaling at single-cell resolution following NLR activation remain largely unexplored. In this study, we employed an inducible gene expression system to activate NLR required for cell death 4 (NRC4) and performed time-lapse calcium imaging at single-cell resolution using confocal microscopy. Utilizing GCaMP6 transgenic Nicotiana benthamiana, we show that activation of NRC4 leads to a calcium signal peak before the onset of cell death events. The calcium peak was absent in the nrc2/3/4 mutant but was restored upon NRC4 expression, confirming its dependency on NRC function. Further investigation using subcellular markers revealed that the calcium peak coincides with the appearance of resistosome punctate and several key cytological changes in cell death responses, inducing endoplasmic reticulum fragmentation and the cessation of vesicle trafficking. Our findings offer a high-resolution perspective on resistosome-mediated calcium dynamics and lay the foundation for unraveling the role of calcium influx in coordinating subcellular responses that lead to hypersensitive cell death.