The rapid production of reactive oxygen species (ROS) burst is a conserved signaling output in immunity across kingdoms. In plants, perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) activates the NADPH oxidase RBOHD by hitherto unknown mechanisms. Here, we show that RBOHD exists in complex with the receptor kinases EFR and FLS2, which are the PRRs for bacterial EF-Tu and flagellin, respectively. The plasma-membrane-associated kinase BIK1, which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. BIK1 phosphorylates different residues than calcium-dependent protein kinases, and both PAMP-induced BIK1 activation and BIK1-mediated phosphorylation of RBOHD are calcium independent. Importantly, phosphorylation of these residues is critical for the PAMP-induced ROS burst and antibacterial immunity. Our study reveals a rapid regulatory mechanism of a plant RBOH, which occurs in parallel with and is essential for its paradigmatic calcium-based regulation.
Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.
Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation.
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