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.

Flagellin perception in Arabidopsis is through recognition of its highly conserved N-terminal epitope (flg22) by flagellin-sensitive 2 (FLS2). Flg22 binding induces FLS2 heteromerization with BRASSINOSTEROID INSENSITIVE 1-associated kinase 1 (BAK1) and their reciprocal activation followed by plant immunity. Here, we report the crystal structure of FLS2 and BAK1 ectodomains complexed with flg22 at 3.06 angstroms. A conserved and a nonconserved site from the inner surface of the FLS2 solenoid recognize the C- and N-terminal segment of flg22, respectively, without oligomerization or conformational changes in the FLS2 ectodomain. Besides directly interacting with FLS2, BAK1 acts as a co-receptor by recognizing the C terminus of the FLS2-bound flg22. Our data reveal the molecular mechanisms underlying FLS2-BAK1 complex recognition of flg22 and provide insight into the immune receptor complex activation.

Pathogen-associated molecular patterns (PAMPs) elicit basal defense responses in plants, and, in turn, pathogens have evolved mechanisms to overcome these PAMP-induced defenses. To suppress immunity, the phytopathogenic bacterium Pseudomonas syringae secretes effector proteins, the biochemical function and virulence targets of which remain largely unknown. We show that HopAI1, an effector widely conserved in both plant and animal bacterial pathogens, inhibits the Arabidopsis mitogen-activated protein kinases (MAPKs) activated by exposure to PAMPs. HopAI1 inactivates MAPKs by removing the phosphate group from phosphothreonine through a unique phosphothreonine lyase activity, which is required for HopAI1 function. The inhibition of MAPKs by HopA1 suppresses two independent downstream events, namely the reinforcement of cell wall defense and transcriptional activation of PAMP response genes. The MAPKs MPK3 and MPK6 physically interact with HopAI1 indicating that they are direct targets of HopAI1. These findings uncover a mechanism by which Pseudomonas syringae overcomes host innate immunity to promote pathogenesis.

Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The Xanthomonas campestris pv. campestris effector AvrAC uridylylates the Arabidopsis PBL2 kinase, and the latter (PBL2UMP) acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo–electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2UMP in an inactive and intermediate state, respectively. The ZAR1LRR domain, compared with animal NLRLRR domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2UMP is exclusively through RKS1, which interacts with ZAR1LRR. PBL2UMP binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs.

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens 1 . LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes 2 , suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.Arabidopsis thaliana (hereafter Arabidopsis) cell-surface LRR receptor kinases (LRR-RKs) and LRR receptor protein (LRR-RP)-SOBIR1 complexes recruit the co-receptor BAK1 and signal through receptor-like cytoplasmic kinases (RLCKs) to elicit PTI 3 . Intracellular coiled-coil (CC)-nucleotide-binding LRR (NLR) or TOLL-INTERLEUKIN 1 RECEP-TOR (TIR)-NLR receptors 4 require ADR1-type and NRG1-type helper NLRs (hNLRs) and the lipase-like EDS1 family proteins EDS1, PAD4 and SAG101 to confer ETI 5,6 . While the defence outputs for PTI and ETI are qualitatively similar 2 , where and how pathways activated in different cell compartments converge remain unclear. Effective plant defence relies on mutual potentiation of PTI and ETI pathways 7,8 , suggesting mechanistic links between these two tiers of the plant immune system. RLCKs PBL30 and PBL31 mediate PTIThe Arabidopsis class VII RLCK (RLCK-VII) BIK1 promotes LRR-RK-mediated PTI but is a negative regulator of LRR-RP-mediated PTI 9 . To identify RLCK-VII members with positive roles in LRR-RP-dependent PTI, we screened an Arabidopsis RLCK-VII transfer DNA mutant library 10 for ethylene production elicited by fungal pg13(At) 11 , oomycete nlp20 and bacterial eMax (which are recognized by RLP42, RLP23 and RLP1, respectively) 3 (Extended Data Fig. 1a). A pbl31 mutant was defective in response to these elicitors compared with wild-type plants (Columbia-0 (Col-0)) (Extended Data Fig. 1a). PBL31 belongs to RLCK-VII subfamily 7, together ...

Plant pattern recognition receptors (PRRs) perceive microbial and endogenous molecular patterns to activate immune signaling. The cytoplasmic kinase BIK1 acts downstream of multiple PRRs as a rate-limiting component, whose phosphorylation and accumulation are central to immune signal propagation. Previous work identified the calcium-dependent protein kinase CPK28 and heterotrimeric G proteins as negative and positive regulators of BIK1 accumulation, respectively. However, mechanisms underlying this regulation remain unknown. Here we show that the plant U-box proteins PUB25 and PUB26 are homologous E3 ligases that mark BIK1 for degradation to negatively regulate immunity. We demonstrate that the heterotrimeric G proteins inhibit PUB25/26 activity to stabilize BIK1, whereas CPK28 specifically phosphorylates conserved residues in PUB25/26 to enhance their activity and promote BIK1 degradation. Interestingly, PUB25/26 specifically target non-activated BIK1, suggesting that activated BIK1 is maintained for immune signaling. Our findings reveal a multi-protein regulatory module that enables robust yet tightly regulated immune responses.

Plants have evolved multiple defense strategies to cope with pathogens, among which plant immune signaling that relies on cell-surface localized and intracellular receptors takes fundamental roles. Exciting breakthroughs were made recently on the signaling mechanisms of pattern recognition receptors (PRRs) and intracellular nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs). This review summarizes the current view of PRRs activation, emphasizing the most recent discoveries about PRRs' dynamic regulation and signaling mechanisms directly leading to downstream molecular events including mitogen-activated protein kinase (MAPK) activation and calcium (Ca 2+ ) burst. Plants also have evolved intracellular NLRs to perceive the presence of specific pathogen effectors and trigger more robust immune responses. We also discuss the current understanding of the mechanisms of NLR activation, which has been greatly advanced by recent breakthroughs including structures of the first full-length plant NLR complex, findings of NLR sensor-helper pairs and novel biochemical activity of Toll/interleukin-1 receptor (TIR) domain.