ABSTRACT
Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.
Subject(s)
Ascomycota/metabolism , Mitochondria/metabolism , Phytochelatins/metabolism , Plants, Genetically Modified/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Catalysis , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiologyABSTRACT
Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [14 C]-acetyl-CoA to oligogalacturonides. Through site-directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cell Wall/metabolism , Disease Resistance/genetics , Pectins/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Ascomycota/pathogenicity , Botrytis/pathogenicity , Cell Wall/chemistry , Cell Wall/genetics , Cellulose/genetics , Cellulose/metabolism , Mutation , Pectins/chemistry , Phylogeny , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/metabolism , Plants, Genetically Modified/geneticsABSTRACT
Mitogen-activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe-associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity. We found that YODA (YDA) - a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning - also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA-YDA) protein show broad-spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor-Like Kinase, regulating both immunity and stomatal patterning. ER-YDA-mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA-YDA plants exhibit altered cell-wall integrity and constitutively express defense-associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA-YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates. Our results suggest that, in addition to stomata development, the ER-YDA pathway regulates an immune surveillance system conferring broad-spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense hormones.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/immunology , Disease Resistance , MAP Kinase Kinase Kinases/metabolism , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity , Body Patterning , Cell Wall/metabolism , Flagellin/pharmacology , Fungi/physiology , Gene Expression Regulation, Plant , Models, Biological , Mutation/genetics , Pathogen-Associated Molecular Pattern Molecules/metabolism , Plant Stomata/growth & development , Signal Transduction , Up-Regulation/geneticsABSTRACT
The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as signal molecules in Arabidopsis (Arabidopsis thaliana), triggering a signaling cascade that shares some similarities to responses to well-known elicitors such as chitooligomers and OGs. However, in contrast to other known PAMPs/DAMPs, cellobiose stimulates neither detectable reactive oxygen species production nor callose deposition. Confirming our idea that both PAMPs and DAMPs are likely to cooccur at infection sites, cotreatments of cellobiose with flg22 or chitooligomers led to synergistic increases in gene expression. Thus, the perception of cellulose-derived oligomers may participate in cell wall integrity surveillance and represents an additional layer of signaling following plant cell wall breakdown during cell wall remodeling or pathogen attack.
Subject(s)
Arabidopsis/metabolism , Cell Wall/metabolism , Cellulose/metabolism , Oligosaccharides/metabolism , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Cell Wall/genetics , Cell Wall/microbiology , Cellobiose/metabolism , Disaccharides/metabolism , Disease Resistance/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Host-Pathogen Interactions , Mutation , Pectins/metabolism , Plant Diseases/genetics , Plant Diseases/microbiology , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Pseudomonas syringae/physiology , Reverse Transcriptase Polymerase Chain Reaction , Seedlings/genetics , Seedlings/metabolism , Seedlings/microbiology , Transcription Factors/geneticsABSTRACT
The (1,3)-ß-glucan callose is a major component of cell wall thickenings in response to pathogen attack in plants. GTPases have been suggested to regulate pathogen-induced callose biosynthesis. To elucidate the regulation of callose biosynthesis in Arabidopsis thaliana, we screened microarray data and identified transcriptional upregulation of the GTPase RabA4c after biotic stress. We studied the function of RabA4c in its native and dominant negative (dn) isoform in RabA4c overexpression lines. RabA4c overexpression caused complete penetration resistance to the virulent powdery mildew Golovinomyces cichoracearum due to enhanced callose deposition at early time points of infection, which prevented fungal ingress into epidermal cells. By contrast, RabA4c(dn) overexpression did not increase callose deposition or penetration resistance. A cross of the resistant line with the pmr4 disruption mutant lacking the stress-induced callose synthase PMR4 revealed that enhanced callose deposition and penetration resistance were PMR4-dependent. In live-cell imaging, tagged RabA4c was shown to localize at the plasma membrane prior to infection, which was broken in the pmr4 disruption mutant background, with callose deposits at the site of attempted fungal penetration. Together with our interactions studies including yeast two-hybrid, pull-down, and in planta fluorescence resonance energy transfer assays, we concluded that RabA4c directly interacts with PMR4, which can be seen as an effector of this GTPase.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Gene Expression Regulation, Plant , Glucans/metabolism , Glucosyltransferases/metabolism , Plant Diseases/immunology , rab GTP-Binding Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , Ascomycota/physiology , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gene Expression , Glucosyltransferases/genetics , Phenotype , Plant Diseases/microbiology , Plant Epidermis/genetics , Plant Epidermis/immunology , Plant Epidermis/physiology , Plant Epidermis/ultrastructure , Plant Immunity , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/physiology , Plant Leaves/ultrastructure , Plants, Genetically Modified , Two-Hybrid System Techniques , rab GTP-Binding Proteins/geneticsABSTRACT
Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.
Subject(s)
Arabidopsis/drug effects , Chitin/pharmacology , Oligosaccharides/pharmacology , Agriculture/methods , Animals , Arabidopsis/genetics , Arabidopsis/growth & development , Biotechnology/methods , Carbon/metabolism , Chitin/chemistry , Crustacea/chemistry , Gene Expression/drug effects , Molecular Weight , Nitrogen/metabolism , Oligosaccharides/chemistry , SoilABSTRACT
Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Despite broad biological importance, the identity of sugar efflux transporters has remained elusive. Using optical glucose sensors, we identified a new class of sugar transporters, named SWEETs, and show that at least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport. Arabidopsis SWEET8 is essential for pollen viability, and the rice homologues SWEET11 and SWEET14 are specifically exploited by bacterial pathogens for virulence by means of direct binding of a bacterial effector to the SWEET promoter. Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is probably targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Glucose/metabolism , Host-Pathogen Interactions/physiology , Membrane Transport Proteins/metabolism , Animals , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Biological Transport/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , HEK293 Cells , Humans , Models, Biological , Oryza/genetics , Oryza/metabolism , Oryza/microbiology , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Xenopus/geneticsABSTRACT
The Arabidopsis penetration resistance 3 (PEN3) ATP binding cassette transporter participates in nonhost resistance to fungal and oomycete pathogens and is required for full penetration resistance to the barley powdery mildew Blumeria graminis f. sp. hordei. PEN3 resides in the plasma membrane and is recruited to sites of attempted penetration by invading fungal appressoria, where the transporter shows strong focal accumulation. We report that recruitment of PEN3 to sites of pathogen detection is triggered by perception of pathogen-associated molecular patterns, such as flagellin and chitin. PEN3 recruitment requires the corresponding pattern recognition receptors but does not require the BAK1 coreceptor. Pathogen- and pathogen-associated molecular pattern-induced focal accumulation of PEN3 and the penetration resistance 1 (PEN1) syntaxin show differential sensitivity to specific pharmacological inhibitors, indicating distinct mechanisms for recruitment of these defense-associated proteins to the host-pathogen interface. Focal accumulation of PEN3 requires actin but is not affected by inhibitors of microtubule polymerization, secretory trafficking, or protein synthesis, and plasmolysis experiments indicate that accumulation of PEN3 occurs outside of the plasma membrane within papillae. Our results implicate pattern recognition receptors in the recruitment of defense-related proteins to sites of pathogen detection. Additionally, the process through which PEN3 is recruited to the host-pathogen interface is independent of new protein synthesis and BFA-sensitive secretory trafficking events, suggesting that existing PEN3 is redirected through an unknown trafficking pathway to sites of pathogen detection for export into papillae.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arabidopsis/metabolism , Arabidopsis/microbiology , Cell Membrane/metabolism , Protein TransportABSTRACT
In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.
Subject(s)
Ascomycota/pathogenicity , Plant Proteins/physiology , Arabidopsis/genetics , Arabidopsis/physiology , Ascomycota/classification , Ascomycota/physiology , Phylogeny , Plants, Genetically Modified , RNA Interference , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A common response by plants to fungal attack is deposition of callose, a (1,3)-ß-glucan polymer, in the form of cell wall thickenings called papillae, at site of wall penetration. While it has been generally believed that the papillae provide a structural barrier to slow fungal penetration, this idea has been challenged in recent studies of Arabidopsis (Arabidopsis thaliana), where fungal resistance was found to be independent of callose deposition. To the contrary, we show that callose can strongly support penetration resistance when deposited in elevated amounts at early time points of infection. We generated transgenic Arabidopsis lines that express POWDERY MILDEW RESISTANT4 (PMR4), which encodes a stress-induced callose synthase, under the control of the constitutive 35S promoter. In these lines, we detected callose synthase activity that was four times higher than that in wild-type plants 6 h post inoculation with the virulent powdery mildew Golovinomyces cichoracearum. The callose synthase activity was correlated with enlarged callose deposits and the focal accumulation of green fluorescent protein-tagged PMR4 at sites of attempted fungal penetration. We observed similar results from infection studies with the nonadapted powdery mildew Blumeria graminis f. sp. hordei. Haustoria formation was prevented in resistant transgenic lines during both types of powdery mildew infection, and neither the salicylic acid-dependent nor jasmonate-dependent pathways were induced. We present a schematic model that highlights the differences in callose deposition between the resistant transgenic lines and the susceptible wild-type plants during compatible and incompatible interactions between Arabidopsis and powdery mildew.
Subject(s)
Arabidopsis/immunology , Arabidopsis/microbiology , Ascomycota/physiology , Disease Resistance/immunology , Glucans/metabolism , Plant Diseases/immunology , Plant Diseases/microbiology , Adaptation, Physiological , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclopentanes/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Genes, Plant/genetics , Green Fluorescent Proteins/metabolism , Models, Biological , Oxylipins/metabolism , Phenotype , Plants, Genetically Modified , Salicylic Acid/metabolism , Time Factors , Transcription, GeneticABSTRACT
At least two components that modulate plant resistance against the fungal powdery mildew disease are ancient and have been conserved since the time of the monocot-dicot split (≈ 200 Mya). These components are the seven transmembrane domain containing MLO/MLO2 protein and the syntaxin ROR2/PEN1, which act antagonistically and have been identified in the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana, respectively. Additionally, syntaxin-interacting N-ethylmaleimide sensitive factor adaptor protein receptor proteins (VAMP721/722 and SNAP33/34) as well as a myrosinase (PEN2) and an ABC transporter (PEN3) contribute to antifungal resistance in both barley and/or Arabidopsis. Here, we show that these genetically defined defense components share a similar set of coexpressed genes in the two plant species, comprising a statistically significant overrepresentation of gene products involved in regulation of transcription, posttranslational modification, and signaling. Most of the coexpressed Arabidopsis genes possess a common cis-regulatory element that may dictate their coordinated expression. We exploited gene coexpression to uncover numerous components in Arabidopsis involved in antifungal defense. Together, our data provide evidence for an evolutionarily conserved regulon composed of core components and clade/species-specific innovations that functions as a module in plant innate immunity.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis , Ascomycota/pathogenicity , Hordeum , Membrane Proteins/genetics , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity/genetics , Regulon , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/microbiology , Arabidopsis Proteins/metabolism , Ascomycota/immunology , Computational Biology , Gene Expression Regulation, Plant , Hordeum/genetics , Hordeum/immunology , Hordeum/microbiology , Membrane Proteins/metabolism , Plants, Genetically ModifiedABSTRACT
Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/microbiology , Botrytis/physiology , Cell Wall/metabolism , Immunity, Innate/immunology , Mutation/genetics , Plant Diseases/immunology , Acetylation , Adaptation, Physiological , Alleles , Arabidopsis/immunology , Arabidopsis Proteins/metabolism , DNA, Bacterial/genetics , Epitopes/immunology , Fungal Proteins/chemistry , Gene Expression Profiling , Gene Expression Regulation, Plant , Glucans/metabolism , Mutagenesis, Insertional/genetics , Mutant Proteins/isolation & purification , Pectins/metabolism , Phylogeny , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Epidermis/cytology , Plant Epidermis/metabolism , Protein Transport , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Xylans/metabolismABSTRACT
Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.
Subject(s)
Arabidopsis Proteins/immunology , Arabidopsis/immunology , Cell Wall/immunology , Fungi/immunology , Hordeum/immunology , Membrane Proteins/immunology , Membrane Proteins/metabolism , Vesicular Transport Proteins , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cell Wall/microbiology , Cloning, Molecular , Hordeum/cytology , Hordeum/genetics , Hordeum/microbiology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Plant Diseases/microbiology , Protein Binding , Qa-SNARE Proteins , SNARE Proteins , Synaptosomal-Associated Protein 25 , Two-Hybrid System TechniquesABSTRACT
We surveyed differential gene expression patterns during early photomorphogenesis in both wild-type and mutant Arabidopsis defective in HY5, an influential positive regulator of the responses of gene expression to a light stimulus, to identify light-responsive genes whose expression was HY5 dependent. These gene-expression data identified light-regulated zinc finger protein 1 (LZF1), a gene encoding a previously uncharacterized C2C2-CO B-box transcriptional regulator. HY5 has positive trans-activating activity toward LZF1 and binding affinity to LZF1 promoter in vivo. HY5 is needed but not sufficient for the induction of LZF1 expression. Anthocyanin content is significantly diminished in lzf1 under far red, which is the most efficient light for the induction of LZF1. The expression of PAP1/MYB75 is elevated in plants overexpressing LZF1, which leads to the hyperaccumulation of anthocyanin in transgenic Arabidopsis. The transition from etioplast to chloroplast and the accumulation of chlorophyll were notably compromised in the lzf1 mutant. We provide molecular evidence that LZF1 influences chloroplast biogenesis and function via regulating genes encoding chloroplast proteins. In the absence of HY5, mutation of LZF1 leads to further reduced light sensitivity for light-regulated inhibition of hypocotyl elongation and anthocyanin and chlorophyll accumulation. Our data indicate that LZF1 is a positive regulator functioning in Arabidopsis de-etiolation.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Gene Expression Regulation, Plant/physiology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Chloroplasts/metabolism , Chloroplasts/ultrastructure , Down-Regulation , Gene Expression Profiling , Light , Mutation , Nuclear Proteins/genetics , Pancreatitis-Associated Proteins , Promoter Regions, Genetic , Protein Binding , Signal Transduction , Transcription Factors/genetics , Up-RegulationABSTRACT
Some receptor-like kinases (RLK) control plant development while others regulate immunity. The Arabidopsis ERECTA (ER) RLK regulates both biological processes. To discover specific components of ER-mediated immunity, a genetic screen was conducted to identify suppressors of erecta (ser) susceptibility to Plectosphaerella cucumerina fungus. The ser1 and ser2 mutations restored disease resistance to this pathogen to wild-type levels in the er-1 background but failed to suppress er-associated developmental phenotypes. The deposition of callose upon P. cucumerina inoculation, which was impaired in the er-1 plants, was also restored to near wild-type levels in the ser er-1 mutants. Analyses of er cell walls revealed that total neutral sugars were reduced and uronic acids increased relative to those of wild-type walls. Interestingly, in the ser er-1 walls, neutral sugars were elevated and uronic acids were reduced relative to both er-1 and wild-type plants. The cell-wall changes found in er-1 and the ser er-1 mutants are unlikely to contribute to their developmental alterations. However, they may influence disease resistance, as a positive correlation was found between uronic acids content and resistance to P. cucumerina. We propose a specific function for ER in regulating cell wall-mediated disease resistance that is distinct from its role in development.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/microbiology , Cell Wall/physiology , Phyllachorales/physiology , Protein Serine-Threonine Kinases/physiology , Receptors, Cell Surface/physiology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Wall/metabolism , Glucans/metabolism , Immunity, Innate , Mutation , Phenotype , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/microbiology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Uronic Acids/metabolismABSTRACT
Because the initial stages of pathogen invasion are often confined to a limited number of host cells, measures of host responses that are averaged over attacked and non-attacked cells provide an unsatisfactory view of these events. To identify the earliest and often transient responses to pathogen attack, there is considerable interest in monitoring the subcellular events that occur specifically in living host cells. Recent improvements in live-cell imaging using fluorescent-tagged markers have expanded the scope of the experiments that can be performed. Changes in the subcellular distribution of organelles and of fluorescently tagged proteins can be monitored in real time in living tissues during pathogen attack, and the dynamic nature of such changes across space and over time can be determined. The application of these sensitive imaging methods has extended earlier observations, made with Nomarski microscopy or inferred from static transmission electron micrographs, about the focal accumulation of subcellular organelles at sites of pathogen attack. In addition, recent experiments have demonstrated the focused accumulation and interaction of specific plant proteins at penetration sites, opening a new window on early host responses and raising questions about the underlying plant processes that sense and direct this marshalling of host resources to block pathogen entry.
Subject(s)
Plant Cells , Plants/microbiology , Fungi/cytology , Fungi/physiology , Plant Diseases/microbiologyABSTRACT
Plants resist attack by haustorium-forming biotrophic and hemi-biotrophic fungi through fortification of the cell wall to prevent penetration through the wall and the subsequent establishment of haustorial feeding structures by the fungus. While the existence of cell wall-based defences has been known for many years, only recently have the molecular components contributing to such defences been identified. Forward genetic screens identified Arabidopsis mutants impaired in penetration resistance to powdery mildew fungi that were normally halted at the cell wall. Several loci contributing to penetration resistance have been identified and a common feature is the striking focal accumulation of proteins associated with penetration resistance at sites of interaction with fungal appressoria and penetration pegs. The focal accumulation of defence-related proteins and the deposition of cell wall reinforcements at sites of attempted fungal penetration represent an example of cell polarization and raise many questions of relevance, not only to plant pathology but also to general cell biology.
Subject(s)
Arabidopsis/metabolism , Arabidopsis/microbiology , Cell Wall/metabolism , Cell Wall/microbiology , Fungi/pathogenicity , Plant Diseases/microbiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Wall/genetics , Fungi/physiology , Plant Diseases/geneticsABSTRACT
BACKGROUND: The hypersensitive necrosis response (HR) of resistant plants to avirulent pathogens is a form of programmed cell death in which the plant sacrifices a few cells under attack, restricting pathogen growth into adjacent healthy tissues. In spite of the importance of this defense response, relatively little is known about the plant components that execute the cell death program or about its regulation in response to pathogen attack. RESULTS: We isolated the edr2-6 mutant, an allele of the previously described edr2 mutants. We found that edr2-6 exhibited an exaggerated chlorosis and necrosis response to attack by three pathogens, two powdery mildew and one downy mildew species, but not in response to abiotic stresses or attack by the bacterial leaf speck pathogen. The chlorosis and necrosis did not spread beyond inoculated sites suggesting that EDR2 limits the initiation of cell death rather than its spread. The pathogen-induced chlorosis and necrosis of edr2-6 was correlated with a stimulation of the salicylic acid defense pathway and was suppressed in mutants deficient in salicylic acid signaling. EDR2 encodes a novel protein with a pleckstrin homology and a StAR transfer (START) domain as well as a plant-specific domain of unknown function, DUF1336. The pleckstrin homology domain binds to phosphatidylinositol-4-phosphate in vitro and an EDR2:HA:GFP protein localizes to endoplasmic reticulum, plasma membrane and endosomes. CONCLUSION: EDR2 acts as a negative regulator of cell death, specifically the cell death elicited by pathogen attack and mediated by the salicylic acid defense pathway. Phosphatidylinositol-4-phosphate may have a role in limiting cell death via its effect on EDR2. This role in cell death may be indirect, by helping to target EDR2 to the appropriate membrane, or it may play a more direct role.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Ascomycota/pathogenicity , Mutation , Salicylic Acid/metabolism , Arabidopsis/metabolism , Arabidopsis/microbiology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Cell Death/genetics , Cell Death/physiology , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Gene Expression Regulation, Plant , Glucans/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hydrogen Peroxide/metabolism , Microscopy, Confocal , Phenotype , Phosphatidylinositol Phosphates/metabolism , Plant Diseases/genetics , Plant Diseases/microbiology , Plants, Genetically Modified , Protein Binding , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
Deposition of cell wall-reinforcing papillae is an integral component of the plant immune response. The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter plays a role in defense against numerous pathogens and is recruited to sites of pathogen detection where it accumulates within papillae. However, the trafficking pathways and regulatory mechanisms contributing to recruitment of PEN3 and other defenses to the host-pathogen interface are poorly understood. Here, we report a confocal microscopy-based screen to identify mutants with altered localization of PEN3-GFP after inoculation with powdery mildew fungi. We identified a mutant, aberrant localization of PEN3 3 (alp3), displaying accumulation of the normally plasma membrane (PM)-localized PEN3-GFP in endomembrane compartments. The mutant was found to be disrupted in the P4-ATPase AMINOPHOSPHOLIPID ATPASE 3 (ALA3), a lipid flippase that plays a critical role in vesicle formation. We provide evidence that PEN3 undergoes continuous endocytic cycling from the PM to the trans-Golgi network (TGN). In alp3, PEN3 accumulates in the TGN, causing delays in recruitment to the host-pathogen interface. Our results indicate that PEN3 and other defense proteins continuously cycle through the TGN and that timely exit of these proteins from the TGN is critical for effective pre-invasive immune responses against powdery mildews.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Membrane/metabolism , Host-Pathogen Interactions , Plant Diseases/genetics , Plant Diseases/microbiology , Protein Transport/genetics , Protein Transport/physiology , trans-Golgi Network/genetics , trans-Golgi Network/metabolismABSTRACT
The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3.