RESUMEN
Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Inmunidad de la Planta/inmunología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Arabidopsis/enzimología , Endocitosis , Ligandos , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
Asunto(s)
Arabidopsis , Vesículas Cubiertas por Clatrina , Arabidopsis/genética , Arabidopsis/metabolismo , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Proteoma/metabolismo , Proteómica , Factor de Transcripción AP-1/análisis , Factor de Transcripción AP-1/metabolismoRESUMEN
In eukaryotes, dynamins and dynamin-related proteins (DRPs) are high-molecular weight GTPases responsible for mechanochemical fission of organelles or membranes. Of the six DRP subfamilies in Arabidopsis thaliana, AtDRP1 and AtDRP2 family members serve as endocytic accessory proteins in clathrin-mediated endocytosis. Most studies have focused on AtDRP1A and AtDRP2B as critical modulators of plant pattern-triggered immunity (PTI) against pathogenic, flagellated Pseudomonas syringae pv. tomato DC3000 bacteria and immune signaling in response to the bacterial flagellin peptide flg22. Much less is known about AtDRP2A, the closely related paralog of AtDRP2B. AtDRP2A and AtDRP2B are the only classical, or bona fide, dynamins in Arabidopsis, based on their evolutionary conserved domain structure with mammalian dynamins functioning in endocytosis. AtDRP2B but not AtDRP2A is required for robust ligand-induced endocytosis of the receptor kinase FLAGELLIN SENSING2 for dampening of early flg22 signaling. Here, we utilized Arabidopsis drp2a null mutants to identify AtDRP2A as a positive contributor to effective PTI against P. syringae pv. tomato DC3000 bacteria, consistent with reduced PATHOGEN RELATED1 (PR1) messenger RNA accumulation. We provide evidence that AtDRP2A is a novel modulator of late flg22 signaling, contributing positively to PR1 gene induction but negatively to polyglucan callose deposition. AtDRP2A has no apparent roles in flg22-elicited mitogen-activated protein kinase defense marker gene induction. In summary, this study adds the evolutionary conserved dynamin AtDRP2A to a small group of vesicular trafficking proteins with roles as non-canonical contributors in immune responses, likely due to modulating one or both the localization and activity of multiple different proteins with distinct contributions to immune signaling. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pseudomonas syringae/fisiología , Flagelina , Bacterias/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Dinaminas/farmacología , Inmunidad de la Planta , Regulación de la Expresión Génica de las PlantasRESUMEN
Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Dinaminas/metabolismo , Flagelina/metabolismo , Inmunidad de la Planta/fisiología , Pseudomonas syringae/patogenicidad , Endocitosis/efectos de los fármacosRESUMEN
The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Inmunidad de la Planta/genética , Inmunidad de la Planta/fisiología , Proteínas Quinasas/genética , Pseudomonas syringae/patogenicidad , Transducción de Señal/genética , Transducción de Señal/fisiología , Red trans-Golgi/metabolismoRESUMEN
Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.
Asunto(s)
Arabidopsis/fisiología , Proteínas de Unión al GTP/deficiencia , Inmunidad Innata/fisiología , Inmunidad de la Planta/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/fisiología , Flagelina/inmunología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Mutación/genética , NADPH Oxidasas/fisiología , Proteínas Quinasas/inmunología , Transducción de SeñalRESUMEN
FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogen-activated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (>2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flagelina/metabolismo , Proteínas Quinasas/metabolismo , Androstadienos/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Flagelina/farmacología , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Tirfostinos/farmacología , WortmaninaRESUMEN
Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dinaminas/metabolismo , Proteínas de Unión al GTP/metabolismo , Células Germinativas de las Plantas/crecimiento & desarrollo , Mitosis , Arabidopsis/embriología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Dinaminas/genética , Proteínas de Unión al GTP/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Células Germinativas de las Plantas/ultraestructura , Mutagénesis Insercional , Mutación , Polen/crecimiento & desarrollo , Polen/ultraestructura , Transducción de SeñalRESUMEN
In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Pseudomonas syringae , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Inmunidad de la Planta/fisiología , Clatrina/metabolismoRESUMEN
Arabidopsis DYNAMIN-RELATED PROTEIN1A (AtDRP1A) and AtDRP2B are large GTPases that function together in endocytosis for effective cytokinesis, cell enlargement and development. A recent study shows that these DRPs contribute to ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (AtFLS2) to modulate flg22-immune signaling, and they are required for immunity against Pseudomonas syringae pv. tomato bacteria. Here, we demonstrate that atdrp1a and atdrp2b single mutants showed increased susceptibility to Botrytis cinerea indicating that AtDRP1A and AtDRP2B are necessary for effective resistance against this necrotrophic fungus. Thus, we expanded our limited understanding of clathrin endocytic accessory proteins in immunity against plant pathogens.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Micosis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis , Clatrina/metabolismo , Clatrina/farmacología , Dinaminas/genética , Dinaminas/metabolismo , Flagelina/farmacología , Regulación de la Expresión Génica de las Plantas , Ligandos , Enfermedades de las Plantas/microbiología , Pseudomonas syringaeRESUMEN
Eukaryotes employ complex immune mechanisms for protection against microbial pathogens. Here, we identified SCD1 (Stomatal Cytokinesis-Defective 1), previously implicated in growth and development through its role in cytokinesis and polarized cell expansion (Falbel, T. G., Koch, L. M., Nadeau, J. A., Segui-Simarro, J. M., Sack, F. D., and Bednarek, S. Y. (2003) Development 130, 4011-4024) as a novel component of innate immunity. In Arabidopsis, SCD1 is a unique gene encoding for the only protein containing a complete DENN (Differentially Expressed in Normal and Neoplastic cells) domain. The DENN domain is a largely uncharacterized tripartite protein motif conserved among eukaryotic proteins. We show that conditional scd1-1 plants containing a point mutation in a conserved DENN residue affected a subset of signaling responses to some bacterial pathogen-associated molecular patterns (PAMPs). Consistent with increased transcript accumulation of Pathogen-related (PR) genes, scd1-1 plants were more resistant to Pseudomonas syringae pathovar tomato (Pst) DC3000 infection implicating SCD1 as a negative regulator of basal resistance against bacteria. scd1-1 plants were different from known mutants exhibiting constitutive expressor of PR (cpr)-like phenotypes, in that growth impairment of scd1-1 plants was genetically independent of constitutive immune response activation. For scd1-1, shift to elevated temperature or introduction of a mutant allele in Salicylic acid Induction-Deficient 2 (SID2) suppressed constitutive defense response activation. sid2-2 also repressed the resistance phenotype of scd1-1. Temperature shift and sid2-2, however, did not rescue conditional growth and sterility defects of scd1-1. These results implicate SCD1 in multiple cellular pathways, possibly by affecting different proteins. Overall, our studies identified a novel role for eukaryotic DENN proteins in immunity against bacteria.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Inmunidad Innata , Proteínas de Plantas/metabolismo , Estomas de Plantas/inmunología , Estomas de Plantas/metabolismo , Plantas/inmunología , Plantas/microbiología , Agrobacterium tumefaciens/fisiología , Secuencia de Aminoácidos , Eliminación de Gen , Datos de Secuencia Molecular , Fenotipo , Desarrollo de la Planta , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estomas de Plantas/crecimiento & desarrollo , Estomas de Plantas/microbiología , Plantas/metabolismo , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/fisiología , Especies Reactivas de Oxígeno/metabolismo , TemperaturaRESUMEN
Callose is a ß-1,3-glucan polysaccharide that is deposited at discrete sites in the plant cell wall in response to microbial pathogens, likely contributing to protection against pathogen infection. Increased callose deposition also occurs in response to the 22-amino acid peptide flg22, a pathogen-associated molecular pattern (PAMP) derived from bacterial flagellin protein. Here, we provide protocols for callose staining using aniline blue in cotyledon and leaf tissue of the model plant Arabidopsis thaliana. Aniline blue stain utilizes a fluorochrome that complexes with callose for its visualization by microscopy using an ultraviolet (UV) filter. For robust quantification of callose deposits, we outline an automated image analysis workflow utilizing the freely available Fiji (Fiji Is Just ImageJ; NIH) software and a Trainable Weka Segmentation (TWS) plugin. Our methodology for automated analysis of large batches of images can be easily adapted to quantify callose in other tissues and plant species, as well as to quantify fluorescent structures other than callose.
Asunto(s)
Arabidopsis/metabolismo , Cotiledón/metabolismo , Glucanos/metabolismo , Procesamiento de Imagen Asistido por Computador , Hojas de la Planta/metabolismo , Coloración y Etiquetado/métodos , Compuestos de Anilina/metabolismo , Automatización , Flagelina/metabolismoRESUMEN
The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner by plant Cf genes that encode membrane-anchored extracytoplasmic leucine-rich repeat (LRR) glycoproteins, which confer recognition of their cognate fungal avirulence (Avr) proteins. Cf-9 and Cf-4 are two such proteins that are 91% identical yet recognize the sequence-unrelated fungal avirulence determinants Avr9 and Avr4, respectively. As shown previously, Cf-4 specificity is determined by three putative solvent-exposed residues in the central LRR and a deletion of two LRR relative to Cf-9. In this study, we focused on identifying the specificity determinants of Cf-9. We generated chimeras between Cf-9 and its close homologue Cf-9B and identified five amino acid residues that constitute major specificity determinants of Cf-9. Introduction of these residues into Cf-9B allowed recognition of Avr9. Consistent with a role in recognition specificity, the identified residues are putatively solvent exposed in the central LRR and occupy hypervariable positions in the global Cf alignment. One of the specificity residues is not found in any other known Cf protein, suggesting the importance of diversifying selection rather than sequence exchange between homologues. Interestingly, there is an overlap between the Cf-4 and Cf-9 specificity-determining residues, precluding a protein with dual specificity.
Asunto(s)
Cladosporium/patogenicidad , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Secuencia de Aminoácidos , Aminoácidos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes de Plantas , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Proteínas Repetidas Ricas en Leucina , Solanum lycopersicum/fisiología , Glicoproteínas de Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/fisiología , Proteínas/química , Proteínas/genética , Proteínas/fisiología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , SolventesRESUMEN
At the host-pathogen interface, the protein composition of the plasma membrane (PM) has important implications for how a plant cell perceives and responds to invading microbial pathogens. A plant's ability to modulate its PM composition is critical for regulating the strength, duration, and integration of immune responses. One mechanism by which plant cells reprogram their cell surface is vesicular trafficking, including secretion and endocytosis. These trafficking processes add or remove cargo proteins (such as pattern-recognition receptors, transporters, and other proteins with immune functions) to or from the PM via small, membrane-bound vesicles. Clathrin-coated vesicles (CCVs) that form at the PM and trans-Golgi network/early endosomes have emerged as the prominent vesicle type in the regulation of plant immune responses. In this review, we discuss the roles of the CCV core, adaptors, and accessory components in plant defense signaling and immunity against various microbial pathogens.
Asunto(s)
Vesículas Cubiertas por Clatrina , Clatrina , Endocitosis , Inmunidad de la Planta , Transducción de SeñalRESUMEN
In plants, the trans-Golgi network (TGN) functionally overlaps with the early endosome (EE), serving as a central sorting hub to direct newly synthesized and endocytosed cargo to the cell surface or vacuole. Here, we focus on the emerging role of the TGN/EE in sorting of immune cargo proteins for effective plant immunity against pathogenic bacteria and fungi. Specific vesicle coat and regulatory components at the TGN/EE ensure that immune cargoes are correctly sorted and transported to the location of their cellular functions. Our understanding of the identity of immune cargoes and the underlying cellular mechanisms regulating their sorting are still rudimentary, but this knowledge is essential to understanding the physiological contribution of the TGN/EE to effective immune responses.
Asunto(s)
Endosomas/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Red trans-Golgi/metabolismo , Transporte de ProteínasRESUMEN
Plants are equipped with a suite of plant pattern recognition receptors (PRRs) that must be properly trafficked to and from the plasma membrane (PM), which serves as the host-pathogen interface, for robust detection of invading pathogenic microbes. Recognition of bacterial flagellin, or the derived peptide flg22, is facilitated by the PM-localized PRR, FLAGELLIN SENSING 2 (FLS2). Upon flg22 binding, FLS2 is rapidly internalized from the PM into endosomal compartments and subsequently degraded. To understand better the integration of FLS2 endocytosis and signaling outputs, we developed methods for the quantitative analysis of FLS2 trafficking using freely available bioimage informatic tools. Emphasis was placed on robust recognition of features and ease of access for users. Using the free and open-source software Fiji (Fiji is just ImageJ) and Trainable Weka Segmentation (TWS) plug-in, we developed a workflow for the automated identification of green fluorescent protein (GFP)-tagged FLS2 in endosomal puncta. Fiji-TWS methods can be adapted with ease for the analysis of FLS2 trafficking in various genetic backgrounds as well as for the endocytic regulation of diverse plant PRRs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas Quinasas/metabolismo , Proteínas de Arabidopsis/química , Automatización , Membrana Celular/metabolismo , Endocitosis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ligandos , Proteínas Quinasas/química , Proteolisis , Programas InformáticosRESUMEN
The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.
Asunto(s)
Proteínas de Arabidopsis/aislamiento & purificación , Arabidopsis/química , Fraccionamiento Celular/métodos , Membrana Celular/química , Proteínas de la Membrana/aislamiento & purificación , Plantones/química , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Centrifugación/instrumentación , Centrifugación/métodos , Cetomacrogol/química , Microsomas/química , Microsomas/metabolismo , Células Vegetales/química , Células Vegetales/metabolismo , Plantones/metabolismo , Tensoactivos/químicaRESUMEN
Callose deposition within the cell wall is a well-documented plant immune response to pathogenic organisms as well as to pathogen-/microbe- associated molecular patterns (P/MAMPs). However, the molecular mechanisms that modulate pathogen-induced callose deposition are less understood. We reported previously that Arabidopsis plants lacking the vesicle trafficking component DYNAMIN-RELATED PROTEIN 2B (DRP2B) display increased callose deposition in response to the PAMP flg22. Here, we show that increased number of flg22-induced callose deposits in drp2b leaves is fully dependent on the callose synthase POWDERY MILDEW RESISTANT 4 (PMR4). We propose that in addition to functioning in flg22-induced endocytosis of the plant receptor, FLAGELLIN SENSING 2, DRP2B may regulate the trafficking of proteins involved in callose synthesis, such as PMR4, and/or callose degradation.
Asunto(s)
Arabidopsis/metabolismo , Glucanos/metabolismo , Proteínas de Arabidopsis/metabolismo , Ascomicetos/fisiología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/metabolismo , Enfermedades de las Plantas , Proteínas Quinasas/metabolismoRESUMEN
Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane-derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co-immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc.
RESUMEN
In the model plant Arabidopsis, the best studied Pattern-triggered immunity (PTI) system is perception of the bacterial pathogen-associated molecular pattern (PAMP) flagellin, or its active peptide-derivative flg22, by the plasma membrane-localized receptor FLAGELLIN SENSING 2 (FLS2). Flg22 perception initiates an array of immune responses including the fast and transient production of reactive oxygen species (ROS). In addition, FLS2 undergoes ligand-induced endocytosis and subsequent degradation within 60 min of flg22-treatment. Luminol-based assays are routinely used to measure extracellular ROS production within minutes after flg22 treatment. Many mutants in flg22-response pathways display defects in flg22-induced ROS production. Here, we describe a luminol-based ROS Re-elicitation Assay that can be utilized to quantitatively assess flg22-signaling competency of FLS2 at times during which FLS2 is internalized, trafficked through endosomal compartments, and degraded in response to flg22. This assay may also be employed to correlate FLS2 signaling competency with receptor accumulation in vesicular trafficking mutants that either affect FLS2 endocytosis or replenishment of FLS2 through the secretory pathway. In addition, this assay can be extended to studies of other PAMP (ligand)-receptor pairs.