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...

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.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

Plants have evolved a large number of receptor-like cytoplasmic kinases (RLCKs) that often functionally and physically associate with receptor-like kinases (RLKs) to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1-and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.