Proteomics and phosphoproteomics revealed molecular networks of stomatal immune responses.

Dynamic protein and phosphoprotein profiles uncovered the overall regulation of stomata movement against pathogen invasion and phosphorylation states of proteins involved in ABA, SA, calcium and ROS signaling, which may modulate the stomatal immune response. Stomatal openings represent a major route of pathogen entry into the plant, and plants have evolved mechanisms to regulate stomatal aperture as innate immune response against bacterial invasion. However, the mechanisms underlying stomatal immunity are not fully understood. Taking advantage of high-throughput liquid chromatography mass spectrometry (LC-MS), we performed label-free proteomic and phosphoproteomic analyses of enriched guard cells in response to a bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In total, 495 proteins and 1229 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, including abscisic acid and salicylic acid hormone signaling, calcium and reactive oxygen species signaling. We also showed that dynamic changes of phosphoprotein WRKY transcription factors may play a crucial role in regulating stomata movement in plant immunity. The identified proteins/phosphoproteins and the pathways form interactive molecular networks to regulate stomatal immunity. This study has provided new insights into the multifaceted mechanisms of stomatal immunity. The differential proteins and phosphoproteins are potential targets for engineering or breeding of crops for enhanced pathogen defense.