RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis

Stomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H2O2) signatures in guard cells are poorly understood. We used the H2O2 sensor roGFP2-Orp1 and a ROS-specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over-oxidation of roGFP2-Orp1 by a pathogen-associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2-Orp1 oxidation. In contrast, RBOHF was necessary for PAMP-mediated ROS production measured by a fluorescein-based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H2O2-mediated stomatal closure at 100 mu M while higher H2O2 concentration up to 1 mM did not promote stomatal closure in wild-type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.