Real‐time monitoring of subcellular H2O2 dynamics by genetically encoded probe roGFP2‐PRXIIB

ABSTRACT In plants, genetically encoded probes based on redox‐sensitive green fluorescent protein (roGFP) have been used to detect hydrogen peroxide (H 2 O 2 ) levels by fusing exogenous thiol peroxidases, such as Orp1 and Tsa2. However, the effectiveness of these thiol peroxidases compared to endogenous ones remains unexplored. Here, we develop a H 2 O 2 probe by fusing roGFP2 to an endogenous H 2 O 2 sensor, type II peroxiredoxin (PRXIIB), which displayed enhanced responsiveness and conversion kinetics compared to roGFP2‐Orp1 in vitro and superior sensitivity to H 2 O 2 in vivo . The roGFP2‐PRXIIB probe allowed robust visualization of H 2 O 2 production in abiotic and biotic stresses, and growing pollen tubes. We further targeted roGFP2‐PRXIIB to cytosol, nuclei, mitochondria and chloroplasts to monitor H 2 O 2 accumulation in real time in different subcellular compartments during immune activation, and the analyses revealed different temporal patterns of H 2 O 2 accumulation during pattern‐ and effector‐triggered immune responses in different compartments. Taken together, the work provides an ultra‐sensitive probe for H 2 O 2 dynamics in diverse plant biological processes.