Investigating Tools to Study Calcium Dynamics in Arabidopsis CPK mutant Immune Response

Pattern-triggered immunity (PTI) is an ancient, conserved branch of the plant immune system activated by pathogen-associated molecular patterns. Calcium signaling is an important part of the immune pathway and plant responses to stress, yet the spatiotemporal dynamics of calcium during PTI are not well understood. Previous research has demonstrated the role of calcium-dependent protein kinases (CPKs) in downstream cytoskeletal regulation and actin reorganization, but the role of calcium in these pathways is unknown. Here, we validate tools to study calcium dynamics using the model plant system Arabidopsis thaliana, and mutations in CPK3-2 and CPK6, which have previously shown cytoskeletal phenotypes during PTI. T-DNA insertional knockout lines cpk3-2 and cpk6 were used to explore the connection between CPK pathways, calcium signaling, and the cytoskeletal PTI response induced by the bacterial flagellum-derived protein flg22. The knockouts were crossed with the genetically encoded calcium biosensor RGECO1/mTurquoise. Seed lines were bulked and genotyped to select homozygote lines for the T–DNA insertional mutant, as well as screened via confocal microscopy to check the fluorescence and functionality of the RGECO1/mTurquoise cassette. Time-lapse images were taken of selected plants with the confocal microscope after inoculation with flg22, and their initial calcium responses were compared with the wildtype RGECO1 cross. The preliminary results presented here give further insight into the roles CPK and calcium play in the PTI response, and how best to continue studying calcium and cytoskeletal dynamics in plant cells.