Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucinerich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr 237 of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/ BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling.pathogen-associated molecular pattern/microbe-associated molecular pattern-triggered immunity | phosphorylation | pattern recognition receptor | BRI1-associated receptor kinase | flagellin sensing 2 P lants and animals live in an environment with a diverse array of microorganisms and have developed the capacity to timely detect potential infectious agents without destroying their own tissues. Innate immunity, the first line of inducible defense, is triggered instantaneously upon the detection of conserved pathogen-or microbe-associated molecular patterns (PAMP/ MAMPs) (1-5). In plants, MAMPs are usually perceived by cellsurface pattern-recognition receptors (PRRs) and mount PAMP/ MAMP-triggered immunity (PTI). Different MAMPs likely trigger convergent immune signaling events, including changes in cytoplasmic Ca 2+ levels, activation of MAP kinase (MAPK) cascades, induction of defense-related genes, production of reactive oxygen species and nitric oxide, deposition of callose to reinforce the cell wall, and stomatal closure to prevent pathogen entry (1-5). PTI is important for plants to thwart off a broad spectrum of potential pathogens.One of the best-characterized plant MAMP receptors is the leucine-rich repeat receptor kinase (LRR-RK) protein FLS2 that recognizes a conserved 22-amino-acid peptide (flg22) from bacterial flagellin (6). Upon flagellin perception, FLS2 rapidly associates with another LRR-receptor-like kinase (RLK), BAK1, thereby initiating downstream signaling (7,8). BAK1 was originally identified as a BRI1-associated receptor kinase mediating brassinosteroid signaling (9, 10). Brassinosteroids (BRs), a class of plant hormone with essential roles in plant growth and development, are perceived by LRR-RK BRI1, ...

Innate immunity represents the first line of inducible defense against microbial infection in plants and animals1–3. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), such as flagellin, initiates convergent signalling pathways involving MAP kinase (MAPK) cascades and global transcriptional changes to boost immunity1–4. Although Ca2+ has long been recognized as an essential and conserved primary mediator in plant defense responses, how Ca2+ signals are sensed and relayed into early MAMP signalling is unknown5,6. Here, we use a functional genomic screen and genome-wide gene expression profiling to show that four calcium-dependent protein kinases (CDPKs) are Ca2+ sensor PKs critical to transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in synthesis of defense peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by flg22, as well as compromised pathogen defense. In contrast to negative roles of calmodulin (CAM) and a CAM-activated transcription factor in plant defense7,8, the present study reveals Ca2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLS2 senses bacterial flagellin and initiates immune signaling by association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases PUB12 and PUB13 to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12/13 and is required for FLS2-PUB12/13 association. PUB12/13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.

SUMMARY Successful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes.

Plants and animals possess innate immune systems to prevent infections and are effectively "nonhosts" for most potential pathogens. The molecular mechanisms underlying nonhost immunity in plants remain obscure. In Arabidopsis, nonhost/nonpathogenic Pseudomonas syringae sustains but pathogenic P. syringae suppresses early MAMP (microbe-associated molecular pattern) marker-gene activation. We performed a cell-based genetic screen of virulence factors and identified AvrPto and AvrPtoB as potent and unique suppressors of early-defense gene transcription and MAP kinase (MAPK) signaling. Unlike effectors of mammalian pathogens, AvrPto and AvrPtoB intercept multiple MAMP-mediated signaling upstream of MAPKKK at the plasma membrane linked to the receptor. In transgenic Arabidopsis, AvrPto blocks early MAMP signaling and enables nonhost P. syringae growth. Deletions of avrPto and avrPtoB from pathogenic P. syringae reduce its virulence. The studies reveal a fundamental role of MAMP signaling in nonhost immunity, and a novel action of type III effectors from pathogenic bacteria.

Pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) are detected as nonself by host pattern recognition receptors (PRRs) and activate pattern-triggered immunity (PTI). Microbial invasions often trigger the production of host-derived endogenous signals referred to as danger-or damage-associated molecular patterns (DAMPs), which are also perceived by PRRs to modulate PTI responses. Collectively, PTI contributes to host defense against infections by a broad range of pathogens. Remarkable progress has been made toward demonstrating the cellular and physiological responses upon pattern recognition, elucidating the molecular, biochemical, and genetic mechanisms of PRR activation, and dissecting the complex signaling networks that orchestrate PTI responses. In this review, we present an update on the current understanding of how plants recognize and respond to nonself patterns, a process from which the seemingly chaotic responses form into a harmonic defense.

SUMMARY Plants use cell surface-resident receptor-like kinases (RLKs) to sense diverse extrinsic and intrinsic cues and elicit distinct biological responses. In Arabidopsis, the ERECTA family RLKs recognize EPIDERMAL PATTERNING FACTORS (EPFs) to specify stomatal patterning. However, little is known about the molecular link between ERECTA activation and intracellular signaling. We report here that the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family RLKs regulate stomatal patterning downstream of EPF ligands and upstream of a MAP kinase cascade. EPF ligands induce the heteromerization of ERECTA and SERK family RLKs. SERKs and ERECTA family RLKs transphosphorylate each other. In addition, SERKs associate with the receptor-like protein (RLP) TMM, a signal modulator of stomata development, in a ligand-independent manner, suggesting that ERECTA, SERKs and TMM form a multi-protein receptorsome consisting of different RLKs and RLP perceiving peptide ligands in regulating stomatal patterning. In contrast to the differential requirement of individual SERK members in plant immunity, cell death control and BR signaling, all four functional SERKs are essential but with unequal genetic contributions to stomatal patterning with descending order of importance from SERK3/BAK1, SERK2, SERK1 to SERK4. Although BR signaling connects stomatal development via multiple components, the function of SERKs in stomatal patterning is uncoupled from their involvement in BR signaling. Our results reveal that the SERK family is a shared key module in diverse Arabidopsis signaling receptorsomes and different combinatorial codes of individual SERK members regulate distinct functions.

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca2+-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.