The Arabidopsis immune receptor FLS2 senses the bacterial flagellin epitope flg22 to activate transient elevation of cytosolic calcium ions, production of reactive oxygen species (ROS), and other signaling events to coordinate antimicrobial defenses, such as stomatal closure that limits bacterial invasion. However, how FLS2 regulates these signaling events remains largely unknown. Here we show that the receptor-like cytoplasmic kinase BIK1, a component of the FLS2 immune receptor complex, not only positively regulates flg22-triggered calcium influx but also directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation. Furthermore, BIK1 and RbohD form a pathway that controls stomatal movement in response to flg22, thereby restricting bacterial entry into leaf tissues. These findings highlight a direct role of the FLS2 complex in the regulation of RbohD-mediated ROS production and stomatal defense.

Plants deploy numerous cell surface-localized pattern-recognition receptors (PRRs) to perceive host- and microbe-derived molecular patterns that are specifically released during infection and activate defense responses. The activation of the mitogen-activated protein kinases MPK3, MPK4, and MPK6 (MPK3/4/6) is a hallmark of immune system activation by all known PRRs and is crucial for establishing disease resistance. The MAP kinase kinase kinase (MAPKKK) MEKK1 controls MPK4 activation, but the MAPKKKs responsible for MPK3/6 activation downstream of diverse PRRs and how the perception of diverse molecular patterns leads to the activation of MAPKKKs remain elusive. Here, we show that two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens in The receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance. Surprisingly, MPK6 further phosphorylates MAPKKK5 Ser-682 and Ser-692 to enhance MPK3/6 activation and disease resistance, pointing to a positive feedback mechanism. Finally, MEKK1 Ser-603 is phosphorylated by both RLCK VII and MPK4, which is required for pattern-triggered MPK4 activation. These findings illustrate central mechanisms by which multiple PRRs activate MAPK cascades and disease resistance.