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 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 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.

SUMMARYCotton is an important cash crop worldwide, and is a significant source of fiber, feed, foodstuff, oil and biofuel products. Considerable effort has been expended to increase sustainable yield and quality through molecular breeding and genetic engineering of new cotton cultivars. Given the recent availability of the whole-genome sequence of cotton, it is necessary to develop molecular tools and resources for large-scale analysis of gene functions at the genome-wide level. We have successfully developed an Agrobacterium-mediated virusinduced gene silencing (VIGS) assay in several cotton cultivars with various genetic backgrounds. The genes of interest were potently and readily silenced within 2 weeks after inoculation at the seedling stage. Importantly, we showed that silencing GhNDR1 and GhMKK2 compromised cotton resistance to the infection by Verticillium dahliae, a fungal pathogen causing Verticillium wilt. Furthermore, we developed a cotton protoplast system for transient gene expression to study gene functions by a gain-of-function approach. The viable protoplasts were isolated from green cotyledons, etiolated cotyledons and true leaves, and responded to a wide range of pathogen elicitors and phytohormones. Remarkably, cotton plants possess conserved, but also distinct, MAP kinase activation with Arabidopsis upon bacterial elicitor flagellin perception. Thus, using gene silencing assays, we have shown that GhNDR1 and GhMKK2 are required for Verticillium resistance in cotton, and have developed high throughput loss-of-function and gain-of-function assays for functional genomic studies in cotton.