Search

Receptor kinase (RK) families process information from small molecules, short peptides, or glycan ligands to regulate core cellular pathways in plants. To date, whether individual plant RKs are capable of processing signals from distinct types of ligands remains largely unexplored. Addressing this requires the discovery of structurally unrelated ligands that engage the same receptor. Here, we focus on FLAGELLIN-SENSING 2 (FLS2), an RK that senses a peptide of bacterial flagellin to activate antibacterial immunity in Arabidopsis. We interrogate >20,000 potential interactions between small molecules and the sensory domain of FLS2 using a large-scale reverse chemical screen. We discover two small molecules that interact with FLS2 in atypical ways. The surrogate ligands weakly activate the receptor to drive a functional antibacterial response channeled via unusual gene expression programs. Thus, chemical probes acting as biased ligands can be exploited to discover unexpected levels of output flexibility in RKs signal transduction., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Flagellin, the protein subunit of the bacterial flagellum, stimulates the innate immune receptor Toll-like receptor 5 (TLR5) after pattern recognition or evades TLR5 through lack of recognition. This binary response fails to explain the weak agonism of flagellins from commensal bacteria, raising the question of how TLR5 response is tuned. Here, we screened abundant flagellins present in metagenomes from human gut for both TLR5 recognition and activation and uncovered a class of flagellin-TLR5 interaction termed silent recognition. Silent flagellins were weak TLR5 agonists despite pattern recognition. Receptor activity was tuned by a TLR5-flagellin interaction distal to the site of pattern recognition that was present in Salmonella flagellin but absent in silent flagellins. This interaction enabled flagellin binding to preformed TLR5 dimers and increased TLR5 signaling by several orders of magnitude. Silent recognition by TLR5 occurred in human organoids and mice, and silent flagellin proteins were present in human stool. These flagellins were produced primarily by the abundant gut bacteria Lachnospiraceae and were enriched in nonindustrialized populations. Our findings provide a mechanism for the innate immune system to tolerate commensal-derived flagellins while remaining vigilant to the presence of flagellins produced by pathogens.

Oligosaccharide fragments of fungal cell wall glycans are important molecular probes for studying both the biology of fungi and fungal infections of humans, animals, and plants. The fungal cell wall contains large amounts of various polysaccharides that are ligands for pattern recognition receptors (PRRs), eliciting an immune response upon recognition. Towards the establishment of a glycan array platform for the identification of new ligands of plant PRRs, tri-, penta-, and heptasaccharide fragments of different cell wall polysaccharides were prepared. Chito- and β-(1→6)-gluco-oligosaccharides were synthesized by automated glycan assembly (AGA), and α-(1→3)- and α-(1→4)-gluco-oligosaccharides were synthesized in solution using a recently reported highly α-selective glycosylation methodology. Incubation of plants with the synthesized oligosaccharides revealed i) length dependence for plant activation by chito-oligosaccharides and ii) β-1,6-glucan oligosaccharides as a new class of glycans capable of triggering plant activation., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. European Journal of Organic Chemistry published by Wiley-VCH GmbH.)

Plant immune responses must be tightly controlled for proper allocation of resources for growth and development. In plants, endogenous signaling peptides regulate developmental and growth-related processes. Recent research indicates that some of these peptides also have regulatory functions in the control of plant immune responses. This classifies these peptides as phytocytokines as they show analogies with metazoan cytokines. However, the mechanistic basis for phytocytokine-mediated regulation of plant immunity remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as phytocytokines in Arabidopsis thaliana. GLV2 signaling enhances sensitivity of plants to elicitation with immunogenic bacterial elicitors and contributes to resistance against virulent bacterial pathogens. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors. RGI mutants show reduced elicitor sensitivity and enhanced susceptibility to bacterial infection. RGI3 forms ligand-induced complexes with the pattern recognition receptor (PRR) FLAGELLIN SENSITIVE 2 (FLS2), suggesting that RGIs are part of PRR signaling platforms. GLV2-RGI signaling promotes PRR abundance independent of transcriptional regulation and controls plant immunity via a previously undescribed mechanism of phytocytokine activity., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

In nature, roots of healthy plants are colonized by multikingdom microbial communities that include bacteria, fungi, and oomycetes. A key question is how plants control the assembly of these diverse microbes in roots to maintain host-microbe homeostasis and health. Using microbiota reconstitution experiments with a set of immunocompromised Arabidopsis thaliana mutants and a multikingdom synthetic microbial community (SynCom) representative of the natural A. thaliana root microbiota, we observed that microbiota-mediated plant growth promotion was abolished in most of the tested immunocompromised mutants. Notably, more than 40% of between-genotype variation in these microbiota-induced growth differences was explained by fungal but not bacterial or oomycete load in roots. Extensive fungal overgrowth in roots and altered plant growth was evident at both vegetative and reproductive stages for a mutant impaired in the production of tryptophan-derived, specialized metabolites ( cyp79b2/b3 ). Microbiota manipulation experiments with single- and multikingdom microbial SynComs further demonstrated that 1) the presence of fungi in the multikingdom SynCom was the direct cause of the dysbiotic phenotype in the cyp79b2/b3 mutant and 2) bacterial commensals and host tryptophan metabolism are both necessary to control fungal load, thereby promoting A. thaliana growth and survival. Our results indicate that protective activities of bacterial root commensals are as critical as the host tryptophan metabolic pathway in preventing fungal dysbiosis in the A. thaliana root endosphere., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The detection of molecular signals derived from other organisms is central to the evolutionary success of plants in the colonization of Earth. The sensory coding of these signals is critical for marshaling local and systemic immune responses that keep most invading organisms at bay. Plants detect immune signals inside and outside their cells using receptors. Here, we focus on receptors that function at the cell surface. We present recent work that expands our understanding of the repertoire of immune signals sensed by this family of receptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is a low-abundance membrane lipid essential for plasma membrane function 1,2 . In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P 2 production is involved in development, immunity and reproduction 3-5 . However, phospholipid synthesis is highly intricate 6 . It is thus likely that steady-state depletion of PI(4,5)P 2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P 2 to increase 7-9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P 2 in plants), a system for the inducible and tunable depletion of PI(4,5)P 2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P 2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P 2 is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P 2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P 2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.

Immune systems restrict microbial pathogens by identifying "non-self" molecules called microbe-associated molecular patterns (MAMPs). It is unclear how immune responses are tuned to or by MAMP diversity present in commensal microbiota. We systematically studied the variability of commensal peptide derivatives of flagellin (flg22), a MAMP detected by plants. We define substantial functional diversity. Most flg22 peptides evade recognition, while others contribute to evasion by manipulating immunity through antagonism and signal modulation. We establish a paradigm of signal integration, wherein the sequential signaling outputs of the flagellin receptor are separable and allow for reprogramming by commensal-derived flg22 epitope variants. Plant-associated communities are enriched for immune evading flg22 epitopes, but upon physiological stress that represses the immune system, immune-activating flg22 epitopes become enriched. The existence of immune-manipulating epitopes suggests that they evolved to either communicate or utilize the immune system for host colonization and thus can influence commensal microbiota community composition., Competing Interests: Declaration of interests J.L.D. is a co-founder of, and shareholder in, AgBiome LLC, a corporation whose goal is to use plant-associated microbes to improve plant productivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Immune systems respond to "non-self" molecules termed microbe-associated molecular patterns (MAMPs). Microbial genes encoding MAMPs have adaptive functions and are thus evolutionarily conserved. In the presence of a host, these genes are maladaptive and drive antagonistic pleiotropy (AP) because they promote microbe elimination by activating immune responses. The role AP plays in balancing the functionality of MAMP-coding genes against their immunogenicity is unknown. To address this, we focused on an epitope of flagellin that triggers antibacterial immunity in plants. Flagellin is conserved because it enables motility. Here, we decode the immunogenic and motility profiles of this flagellin epitope and determine the spectrum of amino acid mutations that drives AP. We discover two synthetic mutational tracks that undermine the detection activities of a plant flagellin receptor. These tracks generate epitopes with either antagonist or weaker agonist activities. Finally, we find signatures of these tracks layered atop each other in natural Pseudomonads., Competing Interests: Declaration of interests J.L.D. is a co-founder of, and shareholder in, AgBiome LLC, a corporation whose goal is to use plant-associated microbes to improve plant productivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Spontaneously arising channels that transport the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The organizational principles of the root immune system are largely uncharacterized. In the February 6, 2020 issue of Cell, Zhou et al. showed roots are built entirely by immunocompetent cells that require distinct instructions to unlock cell-autonomous immune programs. A root cell's developmental identity combined with its spatial distribution fine-tunes immune signal sensitivity, enabling sector-specific immune responses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment 1 . Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides 2 have previously been proposed as ligands for several members of the CrRLK1L family 1 . However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.

The rice immune receptor XA21 is activated by the sulfated microbial peptide required for activation of XA21-mediated immunity X (RaxX) produced by Xanthomonas oryzae pv. oryzae ( Xoo ). Mutational studies and targeted proteomics revealed that the RaxX precursor peptide (proRaxX) is processed and secreted by the protease/transporter RaxB, the function of which can be partially fulfilled by a noncognate peptidase-containing transporter component B (PctB). proRaxX is cleaved at a Gly-Gly motif, yielding a mature peptide that retains the necessary elements for RaxX function as an immunogen and host peptide hormone mimic. These results indicate that RaxX is a prokaryotic member of a previously unclassified and understudied group of eukaryotic tyrosine sulfated ribosomally synthesized, posttranslationally modified peptides (RiPPs). We further demonstrate that sulfated RaxX directly binds XA21 with high affinity. This work reveals a complete, previously uncharacterized biological process: bacterial RiPP biosynthesis, secretion, binding to a eukaryotic receptor, and triggering of a robust host immune response., Competing Interests: The authors declare no conflict of interest.

In plants, cell-surface immune receptors sense molecular non-self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non-self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity. The mc-3-OH-FAs are sensed in a chain length- and hydroxylation-specific manner, with free ( R )-3-hydroxydecanoic acid [( R )-3-OH-C10:0] representing the strongest immune elicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs-including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones-do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.

In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.

In all organisms, major biological processes are controlled by complex protein-protein interactions networks (interactomes), yet their structural complexity presents major analytical challenges. Here, we integrate a compendium of over 4300 phenotypes with Arabidopsis interactome (AI-1 MAIN ). We show that nodes with high connectivity and betweenness are enriched and depleted in conditional and essential phenotypes, respectively. Such nodes are located in the innermost layers of AI-1 MAIN and are preferential targets of pathogen effectors. We extend these network-centric analyses to Cell Surface Interactome (CSI LRR ) and predict its 35 most influential nodes. To determine their biological relevance, we show that these proteins physically interact with pathogen effectors and modulate plant immunity. Overall, our findings contrast with centrality-lethality rule, discover fast information spreading nodes, and highlight the structural properties of pathogen targets in two different interactomes. Finally, this theoretical framework could possibly be applicable to other inter-species interactomes to reveal pathogen contact points.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSI LRR ) that consists of 567 interactions. To demonstrate the power of CSI LRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSI LRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

The root endodermis forms its extracellular diffusion barrier by developing ringlike impregnations called Casparian strips. A factor responsible for their establishment is the SCHENGEN3/GASSHO1 (SGN3/GSO1) receptor-like kinase. Its loss of function causes discontinuous Casparian strips. SGN3 also mediates endodermal overlignification of other Casparian strip mutants. Yet, without ligand, SGN3 function remained elusive. Here we report that schengen2 (sgn2) is defective in an enzyme sulfating peptide ligands. On the basis of this observation, we identified two stele-expressed peptides (CASPARIAN STRIP INTEGRITY FACTORS, CIF1/2) that complement sgn2 at nanomolar concentrations and induce Casparian strip mislocalization as well as overlignification-all of which depend on SGN3. Direct peptide binding to recombinant SGN3 identifies these peptides as SGN3 ligands. We speculate that CIF1/2-SGN3 is part of a barrier surveillance system, evolved to guarantee effective sealing of the supracellular Casparian strip network., (Copyright © 2017, American Association for the Advancement of Science.)

In plants, perception of invading pathogens involves cell-surface immune receptor kinases. Here, we report that the Arabidopsis SITE-1 PROTEASE (S1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity. This inhibition is mediated by the malectin-like receptor kinase FERONIA (FER), which otherwise facilitates the ligand-induced complex formation of the immune receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2) with their co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) to initiate immune signaling. We show that FER acts as a RALF-regulated scaffold that modulates receptor kinase complex assembly. A similar scaffolding mechanism may underlie FER function in other signaling pathways., (Copyright © 2017, American Association for the Advancement of Science.)

Pathway cross-communication cannot be simply tackled by studying isolated signaling systems. Yet understanding how signal transduction pathways attenuate or reinforce each other in vivo is a challenging task. In plants, biosynthesis and signaling of brassinosteroids (BRs) finely regulate growth and defense programs through a complex array of mechanistic and physiological interactions. Conversely, induction of defenses also impacts on the BR biosynthesis at the transcriptional level. In this chapter, we present an experimental framework to study the physiological connection between BR-controlled growth and defenses. We focus on the signaling pathways regulated by the two archetypal cell surface receptors, BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2 (FLS2), to illustrate the signaling nexus of BRs and plant immunity. In Arabidopsis thaliana, these pathways provide one of the very few systems in which the tools and mechanistic details exist to study cross talk at the molecular level.

Extracellular matrices (ECMs) are central to the advent of multicellular life, and their mechanical properties are modulated by and impinge on intracellular signaling pathways that regulate vital cellular functions. High spatial-resolution mapping of mechanical properties in live cells is, however, extremely challenging. Thus, our understanding of how signaling pathways process physiological signals to generate appropriate mechanical responses is limited. We introduce fluorescence emission-Brillouin scattering imaging (FBi), a method for the parallel and all-optical measurements of mechanical properties and fluorescence at the submicrometer scale in living organisms. Using FBi, we showed that changes in cellular hydrostatic pressure and cytoplasm viscoelasticity modulate the mechanical signatures of plant ECMs. We further established that the measured "stiffness" of plant ECMs is symmetrically patterned in hypocotyl cells undergoing directional growth. Finally, application of this method to Arabidopsis thaliana with photoreceptor mutants revealed that red and far-red light signals are essential modulators of ECM viscoelasticity. By mapping the viscoelastic signatures of a complex ECM, we provide proof of principle for the organism-wide applicability of FBi for measuring the mechanical outputs of intracellular signaling pathways. As such, our work has implications for investigations of mechanosignaling pathways and developmental biology., (Copyright © 2016, American Association for the Advancement of Science.)

Background: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships., Results: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses., Conclusions: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

Plants grow while also defending themselves against phylogenetically unrelated pathogens. Because defense and growth are both costly programs, a plant's success in colonizing resource-scarce environments requires tradeoffs between the two. Here, we summarize efforts aimed at understanding how plants use iterative tradeoffs to modulate differential organ growth when defenses are elicited. First, we focus on shoots to illustrate how light, in conjunction with the growth hormone gibberellin (GA) and the defense hormone jasmonic acid (JA), act to finely regulate defense and growth programs in this organ. Second, we expand on the regulation of growth-defense trade-offs in the root, a less well-studied topic despite the critical role of this organ in acquiring resources in an environment deeply entrenched with disparate populations of microbes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Because they are tethered in space, plants have to make the most of their local growth environment. In order to grow in an ever-changing environment, plants constantly remodel their shapes. This adaptive attribute requires the orchestration of complex environmental signals at the cellular and organismal levels. A battery of small molecules, classically known as phytohormones, allows plants to change their body plan by using highly integrated signaling networks and transcriptional cascades. Amongst these hormones, brassinosteroids (BRs), the polyhydroxylated steroid of plants, influence plant responsiveness to the local environment and exquisitely promote, or interfere with, many aspects of plant development. The molecular circuits that wire steroid signals at the cell surface to the promoters of thousands of genes in the nucleus have been defined in the past decade. This review recapitulates how the transduction of BR signals impacts the temporally unfolding programs of plant growth. First, we summarize the paradigmatic BR signaling pathway acting primarily in cellular expansion. Secondly, we describe the current wiring diagram and the temporal dynamics of the BR signal transduction network. And finally we provide an overview of how key players in BR signaling act as molecular gates to transduce BR signals onto other signaling pathways., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Plants must adapt to their environment and require mechanisms for sensing their surroundings and responding appropriately. An expanded family of more than 200 leucine-rich repeat (LRR) receptor kinases (LRR-RKs) transduces fluctuating and often contradictory signals from the environment into changes in nuclear gene expression. Two LRR-RKs, BRASSINOSTEROID INSENSITIVE 1 (BRI1), a steroid receptor, and FLAGELLIN SENSITIVE 2 (FLS2), an innate immune receptor that recognizes bacterial flagellin, act cooperatively to partition necessary growth-defense trade-offs. BRI1 and FLS2 share common signaling components and slightly different activation mechanisms. BRI1 and FLS2 are paradigms for understanding the signaling mechanisms of LRR-containing receptors in plants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Metazoans and plants use pattern recognition receptors (PRRs) to sense conserved microbial-associated molecular patterns (MAMPs) in the extracellular environment. In plants, the bacterial MAMPs flagellin and elongation factor Tu (EF-Tu) activate distinct, phylogenetically related cell surface pattern recognition receptors of the leucine-rich repeat receptor kinase (LRR-RK) family called FLS2 and EF-Tu receptor, respectively. BAK1 is an LRR-RK coreceptor for both FLS2 and EF-Tu receptor. BAK1 is also a coreceptor for the plant brassinosteroid (BR) receptor, the LRR-RK BRI1. Binding of BR to BRI1 primarily promotes cell elongation. Here, we tune the BR pathway response to establish how plant cells can generate functionally different cellular outputs in response to MAMPs and pathogens. We demonstrate that BR can act antagonistically or synergistically with responses to MAMPs. We further show that the synergistic activities of BRs on MAMP responses require BAK1. Our results highlight the importance of plant steroid homeostasis as a critical step in the establishment of plant immunity. We propose that tradeoffs associated with plasticity in the face of infection are layered atop plant steroid developmental programs.

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5 Å resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.

Receptor kinases with leucine-rich repeat (LRR) extracellular domains form the largest family of receptors in plants. In the few cases for which there is mechanistic information, ligand binding in the extracellular domain often triggers the recruitment of a LRR-coreceptor kinase. The current model proposes that this recruitment is mediated by their respective kinase domains. Here, we show that the extracellular LRR domain of BRI1-ASSOCIATED KINASE1 (BAK1), a coreceptor involved in the disparate processes of cell surface steroid signaling and immunity in plants, is critical for its association with specific ligand-binding LRR-containing receptors. The LRRs of BAK1 thus serve as a platform for the molecular assembly of signal-competent receptors. We propose that this mechanism represents a paradigm for LRR receptor activation in plants.

Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases--BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids--were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms.

The cell surface receptor kinase BRASSINOSTEROID-INSENSITIVE-1 (BRI1) is the major receptor for steroid hormones in Arabidopsis. Plants homozygous for loss-of-function mutations in BRI1 display a reduction in the size of vegetative organs, resulting in dwarfism. The recessive bri1-5 mutation produces receptors that do not accumulate to wild-type levels and are retained mainly in the endoplasmic reticulum. We have isolated a dominant suppressor of the dwarf phenotype of bri1-5 plants. We show that this suppression is caused by a second-site mutation in BRI1, bri1-5R1. The bri1-5R1 mutation partially rescues the phenotypes of bri1-5 in many tissues and enhances bri1-5 phenotypes above wild-type levels in several other tissues. We demonstrate that the phenotypes of bri1-5R1 plants are due to both increased cell expansion and increased cell division. To test the mechanism of bri1-5 suppression, we assessed whether the phenotypic suppression in bri1-5R1 was dependent on ligand availability and the integrity of the signaling pathway. Our results indicate that the suppression of the dwarf phenotypes associated with bri1-5R1 requires both BR biosynthesis and the receptor kinase BRI1-ASSOCIATED KINASE-1 (BAK1). Finally, we show that bri1-5R1 partially restores the accumulation and plasma membrane localization of BRI1. Collectively, our results point toward a model in which bri1-R1 compensates for the protein-folding abnormalities caused by bri1-5, restoring accumulation of the receptor and its delivery to the cell surface.

Plants control their size through the action of several phytohormones. One class of growth-promoting hormones is the brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants. Here, we present the Arabidopsis-specific proteins that are the founding members of key BR signaling pathway components found in all plants. The genetic studies that identified these components are unique to Arabidopsis owing to its rapid generation time, sophisticated genetics, and facile transformation protocols, thereby highlighting the importance of a reference plant for understanding fundamental processes in all land plants.

Plants use the coordinated action of several small-molecule hormones to grow and develop optimally in response to a changing environment. Among these hormones are the brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants. BRs bind a small family of leucine-rich repeat receptor kinases at the cell surface, thereby initiating an intracellular signal transduction cascade that results in the altered expression of hundreds of genes.

Pathogen recognition by the plant immune system is governed by structurally related, polymorphic products of disease resistance (R) genes. RAR1 and/or SGT1b mediate the function of many R proteins. RAR1 controls preactivation R protein accumulation by an unknown mechanism. We demonstrate that Arabidopsis SGT1b has two distinct, genetically separable functions in the plant immune system: SGT1b antagonizes RAR1 to negatively regulate R protein accumulation before infection, and SGT1b has a RAR1-independent function that regulates programmed cell death during infection. The balanced activities of RAR1 and SGT1, in concert with cytosolic HSP90, modulate preactivation R protein accumulation and signaling competence.

Plant cells have two defense systems that detect bacterial pathogens. One is a basal defense system that recognizes complex pathogen-associated molecular patterns (PAMPs). A second system uses disease-resistance (R) proteins to recognize type lll effector proteins that are delivered into the plant cell by the pathogen's type III secretion system. Here we show that these two pathways are linked. We find that two Pseudomonas syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the host's basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1 for degradation and phosphorylation, respectively. We find that RIN4 is itself a regulator of PAMP signaling. The R proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems.

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.

The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.

Type III pili deliver effector proteins (virulence factors) from bacterial pathogens to host cells. Plants express disease resistance (R) proteins that respond specifically to a particular type III effector by activating immune responses. We demonstrated previously that two unrelated type III effectors from Pseudomonas syringae target and modify the Arabidopsis RIN4 protein. Here, we show that AvrRpt2, a third, unrelated type III effector, also targets RIN4 and induces its posttranscriptional disappearance. This effect is independent of the presence of RPS2, the Arabidopsis R protein that senses AvrRpt2. RIN4 overexpression inhibits multiple phenotypes associated with AvrRpt2 function. Conversely, disruption of RIN4 results in RPS2-dependent lethality. RPS2 and RIN4 physically associate in the plant. We suggest that RIN4 is the target of the AvrRpt2 virulence function, and that perturbation of RIN4 activates RPS2. Thus, RIN4 is a point of convergence for the activity of at least three unrelated P. syringae type III effectors.