ABSTRACT
Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Membrane , NLR Proteins/genetics , Phospholipids , Plant Diseases , Plant ImmunityABSTRACT
NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.
Subject(s)
14-3-3 Proteins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Hypocotyl/radiation effects , 14-3-3 Proteins/genetics , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Hypocotyl/metabolism , Indoleacetic Acids/metabolism , Light , Phosphorylation , Phototropism/radiation effects , Protein Binding , Protein Domains , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/immunology , Carboxylic Ester Hydrolases/metabolism , DNA-Binding Proteins/metabolism , Plant Immunity , Protein Serine-Threonine Kinases/metabolism , Arabidopsis Proteins/chemistry , Carboxylic Ester Hydrolases/chemistry , DNA-Binding Proteins/chemistry , Protein Domains , Protein Kinases/chemistry , Protein Kinases/metabolism , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolismABSTRACT
Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.
Subject(s)
Arabidopsis/parasitology , Cytotoxins/metabolism , Host Specificity , Phytophthora/metabolism , Plant Diseases/parasitology , Pythium/metabolism , Sphingolipids/metabolism , Toxins, Biological/metabolism , Binding Sites , Crystallography, X-Ray , Cytotoxins/chemistry , Ethylenes/metabolism , Sphingolipids/chemistryABSTRACT
Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which - epsilon - is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development.
Subject(s)
14-3-3 Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/metabolism , Indoleacetic Acids/metabolism , Membrane Transport Proteins/metabolism , Plant Development , Plant Growth Regulators/metabolism , 14-3-3 Proteins/genetics , Arabidopsis/genetics , Gene Silencing , Gene TargetingABSTRACT
The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.
Subject(s)
Brachypodium/metabolism , Indoleacetic Acids/metabolism , Plant Roots/metabolism , Cell Wall/metabolism , Galactans/metabolism , Signal Transduction/physiologyABSTRACT
Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study.
Subject(s)
14-3-3 Proteins/metabolism , Arabidopsis Proteins/metabolism , beta-Fructofuranosidase/metabolism , 14-3-3 Proteins/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA, Bacterial , Fructose/metabolism , Glucose/metabolism , Light , Mutation , Phosphorylation , Plant Roots/genetics , Plant Roots/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Serine/metabolism , beta-Fructofuranosidase/geneticsABSTRACT
PSI1 was identified as a gene that is co-expressed with the phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 in Arabidopsis thaliana. It represents a plant-specific protein family of unknown function with six members in two clades. Clade 1 members PSI1, PSI2 and PSI3 were characterized in this study. All three are nuclear localized. A predicted N-terminal myristoylation site was functionally analyzed. psi1-1 seedlings have shorter roots and hypocotyls. This growth-retarded phenotype was restored by expression of either wildtype PSI1 or PSI1 G2A with a mutated myristate attachment site in the psi1-1 background suggesting that myristate attachment was not essential for PSI1 function. psi2-1 and psi3-1 seedlings have a wildtype phenotype but overexpression of PSI1 or PSI2 promoted seedling growth. PSI2 activity appears to be linked to PSK signaling as psi2-1 and psi2-1 psi3-1 roots are unresponsive to PSK. PSI3 functions in vegetative plant growth synergistic with PSI2. psi3-1 and particularly psi2-1 psi3-1 rosettes are small. Overexpression of PSI3 promoted plant growth indicating that PSI3 is limiting at the vegetative stage. Severe dwarfism of psi2-1 psi3-1 plants results from reduced cell growth and proliferation and premature leaf growth arrest. Plants further display reduced fertility and premature senescence revealing a crucial function of PSI proteins in vegetative growth and reproduction. Psi single and double knock-out plants have less and PSI3ox plants have more starch compared to wt and growth retardation is partially rescued by sucrose. Our studies reveal a crucial function of the nuclear-localized PSI proteins in growth possibly through metabolic control.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/growth & development , Nuclear Proteins/physiology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Cloning, Molecular , Epistasis, Genetic , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Gene Knockout Techniques , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Signal Transduction , Species Specificity , Starch/metabolismABSTRACT
The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarisation of the plasma membrane which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarisation and wall expansion, the activation of the plasma membrane P-type H+-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyse long-distance BL signaling in Arabidopsis seedlings.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Protein Kinases/metabolism , Proton-Translocating ATPases/metabolism , Threonine/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Green Fluorescent Proteins , Phosphorylation , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Protein Kinases/genetics , Proton-Translocating ATPases/geneticsABSTRACT
To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Cell Membrane/physiology , Cell Wall/physiology , Cholestanols/pharmacology , Protein Kinases/metabolism , Steroids, Heterocyclic/pharmacology , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Adenosine Triphosphatases , Arabidopsis/drug effects , Arabidopsis/genetics , Brassinosteroids , Cell Membrane/enzymology , Cell Wall/drug effects , Electrophysiology , Green Fluorescent Proteins/metabolism , Membrane Potentials , Phosphorylation , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Recombinant Fusion Proteins/metabolism , Seedlings/drug effects , Seedlings/growth & development , Seedlings/physiology , Sodium Acetate/pharmacology , Nicotiana/drug effects , Nicotiana/genetics , Nicotiana/physiologyABSTRACT
14-3-3 Dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client's activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1) revealed 14-3-3s to be essential players in floral induction. Such fascinating discoveries, however, can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account recent surveys of the Arabidopsis 14-3-3 interactome.
ABSTRACT
The plant plasma membrane H(+)-ATPase is kept at a low activity level by its C-terminal domain, the inhibitory function of which is thought to be mediated by two regions (region I and II) interacting with cytoplasmic domains essential for the catalytic cycle. The activity of the enzyme is well known to be regulated by 14-3-3 proteins, the association of which requires phosphorylation of the penultimate H(+)-ATPase residue, but can be abolished by phosphorylation of residues close-by. The current knowledge about H(+)-ATPase regulation is briefly summed up here, combined with data that query some of the above statements. Expression of various C-terminal deletion constructs of PMA2, a H(+)-ATPase isoform from Nicotiana plumbaginifolia, in yeast indicates that three regions, which do not correspond to regions I or II, contribute to autoinhibition. Their individual and combined action can be abolished by (mimicking) phosphorylation of three threonine residues located within or close to these regions. With respect to the wild-type PMA2, mimicking phosphorylation of two of these residues increases enzyme activity. However, constitutive activation of wild-type PMA2 requires 14-3-3 association. Altogether, the data suggest that regulation of the plant H(+)-ATPase occurs in progressive steps, mediated by several protein kinases and phosphatases, thus allowing gradual as well as fine-tuned adjustment of its activity. Moreover, mating-based split ubiquitin assays indicate a complex interplay between the C-terminal domain and the rest of the enzyme. Notably, their tight contact does not seem to be the cause of the inactive state of the enzyme.
Subject(s)
Cell Membrane/enzymology , Plant Proteins/metabolism , Proton-Translocating ATPases/antagonists & inhibitors , Proton-Translocating ATPases/metabolism , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , Amino Acid Sequence , Molecular Sequence Data , Phosphorylation , Plant Proteins/genetics , Protein Structure, Tertiary , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Sequence DeletionABSTRACT
Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant , Heat-Shock Proteins/metabolism , Peptide Fragments/metabolism , Plant Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , Heat Shock Transcription Factors , Heat-Shock Proteins/genetics , Heat-Shock Response/physiology , Peptide Fragments/genetics , Plant Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
NEP1-like proteins (NLPs) are secreted proteins from fungi, oomycetes and bacteria, triggering immune responses and cell death in dicotyledonous plants. It has been unclear for a long time, whether NLPs are toxins or triggers of plant immunity. In a recent study we report that NLPs are toxins that exert cytolytic activity on dicotyledonous plants. Mutational analysis revealed a causal link between membrane damaging, cell death inducing and virulence promoting properties of NLPs. Interestingly, also induction of immune responses by NLPs required the same protein fold, providing evidence for damage-induced immunity in plants. Structural similarity to pore forming toxins from marine invertebrates allows the proposal of a model for the mode of NLP interaction with the host's membrane.
ABSTRACT
Members of the eukaryotic 14-3-3 family are highly conserved proteins that have been implicated in the modulation of distinct biological processes by phosphorylation-dependent protein-protein interactions. In plants, 14-3-3 mediated regulation of house-keeping proteins such as nitrate reductase and the plasma membrane localized H(+)-ATPase has been intensely studied. Recent proteome-wide approaches have indicated that the plant 14-3-3 interactome is comparable in size and functional complexity to its animal counterpart and, furthermore, shifted the focus of attention to signal mediators. In this regard, in vivo analyses of certain signaling proteins, such as BRASSINAZOLE-RESISTANT 1, a transcription factor controlling brassinosteroid responsive gene expression, verified an essential role for 14-3-3s in hormonal signal transduction processes.
Subject(s)
14-3-3 Proteins/metabolism , Arabidopsis Proteins/metabolism , Nuclear Proteins/metabolism , Plants/metabolism , Signal Transduction , DNA-Binding Proteins , Gene Expression Regulation, Plant , Plant Growth Regulators/metabolism , Protein Structure, Tertiary , Proteome/metabolismABSTRACT
Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35A resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.
Subject(s)
Algal Proteins/chemistry , Plant Diseases/microbiology , Plants/microbiology , Pythium/pathogenicity , Toxins, Biological/chemistry , Algal Proteins/genetics , Computer Simulation , Crystallography, X-Ray , Models, Chemical , Pectobacterium/pathogenicity , Phytophthora/pathogenicity , Plant Diseases/immunology , Plants/immunology , Protein Conformation , Protein Folding , Toxins, Biological/genetics , VirulenceABSTRACT
Cotylenin A, a fungal metabolite originally described as a cytokinin-like bioactive substance against plants shows differentiation-inducing and anti-tumor activity in certain human cancers. Here, we present the crystal structure of cotylenin A acting on a 14-3-3 regulatory protein complex. By comparison with the closely related, but non-anticancer agent fusicoccin A, a rationale for the activity of cotylenin A in human cancers is presented. This class of fusicoccane diterpenoids are possible general modulators of 14-3-3 protein-protein interactions. In this regard, specificities for individual 14-3-3/target protein complexes might be achieved by varying the substituent pattern of the diterpene ring system. As the different activities of fusicoccin A and cotylenin A in human cancers suggest, hydroxylation of C12 might be a sufficient determinant of structural specificity.
Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Nicotiana/metabolism , Amino Acid Motifs , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Calorimetry , Crystallography, X-Ray , Diterpenes/pharmacology , Glycosides/metabolism , Glycosides/pharmacology , Humans , Models, Molecular , Molecular Sequence Data , Phosphopeptides/chemistry , Protein Binding/drug effects , Protein Multimerization/drug effects , Protein Stability/drug effects , Protein Structure, Secondary , Proton-Translocating ATPases/chemistry , Structure-Activity RelationshipABSTRACT
The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 A resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled protein did not crystallize under the original conditions, a second crystal form was identified that yielded crystals that diffracted to 2.1 A resolution. A multiple-wavelength anomalous dispersion (MAD) experiment was performed at 100 K and all four selenium sites were identified, which allowed solution of the structure.
Subject(s)
Algal Proteins/chemistry , Pythium/metabolism , Algal Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Escherichia coli/metabolism , Selenomethionine/chemistry , Selenomethionine/metabolism , X-Ray DiffractionABSTRACT
Regulatory 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H(+)-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H(+)-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H(+)-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H(+)-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.
Subject(s)
14-3-3 Proteins/chemistry , Membrane Proteins/chemistry , Plant Proteins/chemistry , Proton-Translocating ATPases/chemistry , 14-3-3 Proteins/metabolism , 14-3-3 Proteins/ultrastructure , Amino Acid Motifs , Binding Sites , Cryoelectron Microscopy , Crystallography, X-Ray , Glycosides/chemistry , Glycosides/metabolism , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Models, Biological , Models, Molecular , Mutation , Plant Proteins/metabolism , Plant Proteins/ultrastructure , Proton-Translocating ATPases/metabolism , Proton-Translocating ATPases/ultrastructure , Nicotiana/metabolismABSTRACT
Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.