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.
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.
Maintaining active growth and effective immune responses is often costly for a living organism to survive. Fine-tuning the shared cross-regulators is crucial for metazoans and plants to make a trade-off between growth and immunity. The Arabidopsis regulatory receptor-like kinase BAK1 complexes with the receptor kinases FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. BR homeostasis and signaling unidirectionally modulate FLS2-mediated immune responses at multiple levels. We have shown previously that BIK1, a receptorlike cytoplasmic kinase, is directly phosphorylated by BAK1 and associates with FLS2/BAK1 complex in transducing flagellin signaling. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutant displays various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and transcriptional regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Thus, BIK1 relays the signaling in plant immunity and BR-mediated growth via distinct phosphorylation by BAK1 and BRI1, respectively. Our studies indicate that BIK1 mediates inverse functions in plant immunity and development via dynamic association with specific receptor complexes and differential phosphorylation events.brassinosteroid insensitive 1 | BRI1-associated receptor kinase | flagellin sensing 2 | botrytis-induced kinase 1 | bri1-Ems-Suppressor 1 M etazoans and plants have evolved complex mechanisms to cope with the constant challenges of environmental stresses while maintaining their growth and development. Being sessile and lacking a sophisticated adaptive immune system, plants possess a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) that modulate growth, development, and innate immunity (1). RLKs sense different extrinsic and intrinsic cues through the extracellular domain and mediate diverse signaling events via the kinase domain. Arabidopsis Brassinosteroid Insensitive 1 (BRI1), a leucine-rich repeat (LRR)-receptor kinase perceives the polyhydroxylated growth hormones brassinosteroids (BR) to regulate plant growth and development (2). Despite structural similarity with BRI1, Flagellin Sensing 2 (FLS2) and EFTu Receptor (EFR) recognize microbe-associated molecular pattern (MAMP) flagellin and elongation factor Tu (EF-Tu), respectively, and initiate innate immune signaling to defend against pathogen attacks (3, 4). Apparently, signaling specificity is achieved by s...
Proteins entering the secretory pathway of eukaryotic cells are folded into their native structures in the endoplasmic reticulum (ER). Disruption of protein folding causes ER stress and activates signaling cascades, designated the unfolded protein response (UPR), that restore folding capacity. In mammals and yeast, the protein disulfide isomerases (PDIs) are key protein folding catalysts activated during UPR. However, little is known about the response of PDI genes to UPR in plants. In Arabidopsis thaliana, we identified 12 PDI genes that differed in polypeptide length, presence of signal peptide and ER retention signal, and the number and positions of thioredoxin and transmembrane domains. AtPDI gene expression was investigated in different tissues, in response to chemically induced UPR, and in null mutants of UPR signaling mediators (AtIRE1-2 and AtbZIP60). The expression of six AtPDI genes was significantly up-regulated by UPR and sharply attenuated by the transcription inhibitor, actinomycin D, indicating UPR induced AtPDI gene transcription. AtPDI and BIP2 (Binding protein) gene expression was not affected in the Atire1-2 mutant exposed to UPR, however, the expression of four AtPDI genes was decreased in the Atbzip60 mutant. We proposed that additional UPR signaling factors complement AtbZIP60 in the activation of AtPDI gene expression during ER stress in plants.
Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase-substrate association regulated by phosphorylation.
The sessile plants have evolved a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) to modulate diverse biological processes, including plant innate immunity. Phosphorylation of the RLK/RLCK complex constitutes an essential step to initiate immune signaling. Two Arabidopsis plasma membrane-resident RLKs, flagellin-sensing 2 and brassinosteroid insensitive 1-associated kinase 1 (BAK1), interact with RLCK Botrytis-induced kinase 1 (BIK1) to initiate plant immune responses to bacterial flagellin. BAK1 directly phosphorylates BIK1 and positively regulates plant immunity. Classically defined as a serine/threonine kinase, BIK1 is shown here to possess tyrosine kinase activity with mass spectrometry, immunoblot, and genetic analyses. BIK1 is autophosphorylated at multiple tyrosine (Y) residues in addition to serine/threonine residues. Importantly, BAK1 is able to phosphorylate BIK1 at both tyrosine and serine/ threonine residues. BIK1Y150 is likely catalytically important as the mutation blocks both tyrosine and serine/threonine kinase activity, whereas Y243 and Y250 are more specifically involved in tyrosine phosphorylation. The BIK1 tyrosine phosphorylation plays a crucial role in BIK1-mediated plant innate immunity as the transgenic plants carrying BIK1Y150F, Y243F, or Y250F (the mutation of tyrosine to phenylalanine) failed to complement the bik1 mutant deficiency in immunity. Our data indicate that plant RLCK BIK1 is a nonreceptor dual-specificity kinase and both tyrosine and serine/ threonine kinase activities are required for its functions in plant immune signaling. Together with the previous finding of BAK1 to be autophosphorylated at tyrosine residues, our results unveiled the tyrosine phosphorylation cascade as a common regulatory mechanism that controls membrane-resident receptor signaling in plants and metazoans.
Rice (Oryza sativa L.) is a chilling-sensitive staple crop that originated in subtropical regions of Asia. Introduction of the chilling tolerance trait enables the expansion of rice cultivation to temperate regions. Here we report the cloning and characterization of HAN1, a quantitative trait locus (QTL) that confers chilling tolerance on temperate japonica rice. HAN1 encodes an oxidase that catalyzes the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and fine-tunes the JA-mediated chilling response. Natural variants in HAN1 diverged between indica and japonica rice during domestication. A specific allele from temperate japonica rice, which gained a putative MYB cis-element in the promoter of HAN1 during the divergence of the two japonica ecotypes, enhances the chilling tolerance of temperate japonica rice and allows it to adapt to a temperate climate. The results of this study extend our understanding of the northward expansion of rice cultivation and provide a target gene for the improvement of chilling tolerance in rice.
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