Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

Background: Arabidopsis thaliana and Pseudomonas syringae pathovar tomato (Pto) provide an excellent plant-bacteria model system to study innate immunity. During pattern-triggered immunity (PTI), cognate host receptors perceive pathogen-associated molecular patterns (PAMPs) as non-self molecules. Pto harbors many PAMPs; thus for experimental ease, many studies utilize single synthesized PAMPs such as flg22, a short protein peptide derived from Pseudomonas flagellin. Flg22 recognition by Arabidopsis Flagellin Sensing 2 (FLS2) initiates a plethora of signaling responses including rapid production of apoplastic reactive oxygen species (ROS). Assessing flg22-ROS has been instrumental in identifying novel PAMP-signaling components; but comparably little is known whether in Arabidopsis, ROS is produced in response to intact live Pto and whether this response can be used to dissect genetic requirements of the plant host and live bacterial pathogens in planta. Results: Here, we report of a fast and robust bioassay to quantitatively assess early ROS in Arabidopsis leaves, a tissue commonly used for pathogen infection assays, in response to living bacterial Pto strains. We establish that live Pto elicits a transient and dose-dependent ROS that differed in timing of initiation, amplitude and duration compared to flg22-induced ROS. Our control experiments confirmed that the detected ROS was dependent on the presence of the bacterial cells. Utilizing Arabidopsis mutants previously shown to be defective in flg22-induced ROS, we demonstrate that ROS elicited by live Pto was fully or in part dependent on RbohD and BAK1, respectively. Because fls2 mutants did not produce any ROS, flagellin perception by FLS2 is the predominant recognition event in live Pto-elicited ROS in Arabidopsis leaves. Furthermore using different Pto strains, our in planta results indicate that early ROS production appeared to be independent of the Type III Secretion System. Conclusions: We provide evidence and necessary control experiments demonstrating that in planta, this ROS bioassay can be utilized to rapidly screen different Arabidopsis mutant lines and ecotypes in combination with different bacterial strains to investigate the genetic requirements of a plant host and its pathogen. For future experiments, this robust bioassay can be easily extended beyond Arabidopsis-Pto to diverse plant-pathosystems including crop species and their respective microbial pathogens.