Pluis, M. H.; Abdennour, D.; Mackrill, J. J.; Evangelisti, E.

Ca2+ signaling mediates rapid cellular responses across eukaryotes, but its spatiotemporal dynamics remain largely inaccessible in many genetically less tractable microbial lineages. Oomycete plant pathogens, including Phytophthora species, undergo rapid transitions between motile, encysted, germinating, and invasive stages, yet the organization of Ca2+ dynamics during these transitions is poorly understood. Here, we adapt the genetically encoded ratiometric biosensor MatryoshCaMP8s for in vivo calcium imaging in Phytophthora palmivora. The reporter is stably expressed without major detectable effects on sporulation or virulence and reports rapid ratiometric responses to cold shock and calcimycin/A23187. Using live-cell imaging, we uncover stage-specific Ca2+ dynamics across the pre-infective and early infection cycle. Sporangia approaching zoospore release display spatially heterogeneous Ca2+ transients, newly formed cysts occasionally exhibit Ca2+ transients, and germinating cysts show recurrent Ca2+ transients at the germ tube tip. Similar sharp ratiometric pulses occur during early plant infection, indicating that polarized Ca2+ transients are not restricted to in vitro germination but recur at the host surface. Together, our work establishes live ratiometric calcium imaging in oomycetes, reveals polarized Ca2+ transients as recurrent signatures of developmental transitions and early host colonization, and opens the way to mechanistic dissection of signaling, polarity, and infection in a major group of plant pathogens.