RESUMO
Immune receptors play important roles in the perception of pathogens and initiation of immune responses in both plants and animals. Intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors constitute a major class of receptors in vascular plants. In the Arabidopsis thaliana mutant suppressor of npr1-1, constitutive 1 (snc1), a gain-of-function mutation in the NLR gene SNC1 leads to SNC1 overaccumulation and constitutive activation of defense responses. From a CRISPR/Cas9-based reverse genetics screen in the snc1 autoimmune background, we identified that mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains, can suppress snc1 phenotypes. TC1b does not appear to be a general immune regulator as it is not required for defense mediated by other tested immune receptors. TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented by ACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b impacts snc1 autoimmunity in a unique way. TC1b can form oligomers and localizes to punctate structures of unknown function. The puncta localization of TC1b strictly requires its coiled-coil (CC) domain, whereas the functionality of TC1b requires the four TRAF domains in addition to the CC. Overall, we uncovered the TRAF domain protein TC1b as a novel positive contributor to plant immunity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Imunidade Vegetal , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Fenótipo , Doenças das PlantasRESUMO
The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.
Assuntos
Arabidopsis , Proteínas SNARE , Membrana Celular/metabolismo , Fusão de Membrana , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Tirosina/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismoRESUMO
Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodosRESUMO
Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gß, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gß1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Subunidades beta da Proteína de Ligação ao GTP , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Morte Celular , Quitina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Imunidade Vegetal/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
The unfoldase CDC48 (Cell Division Cycle 48) is highly conserved in eukaryotes, serving as an AAA + ATPase to extract ubiquitinated proteins from large protein complexes and membranes. Although its biochemical properties have been studied extensively in yeast and animal systems, the biological roles and regulations of the plant CDC48s have been explored only recently. Here we describe the identification of a novel E3 ligase from the SNIPER (snc1-influencing plant E3 ligase reverse genetic) screen, which contributes to plant defense regulation by targeting CDC48A for degradation. SNIPER7 encodes an F-box protein and its overexpression leads to autoimmunity. We identified CDC48s as interactors of SNIPER7 through immunoprecipitation followed by mass spectrometry proteomic analysis. SNIPER7 overexpression lines phenocopy the autoimmune mutant Atcdc48a-4. Furthermore, CDC48A protein levels are reduced or stabilized when SNIPER7 is overexpressed or inhibited, respectively, suggesting that CDC48A is the ubiquitination substrate of SCFSNIPER7 . Taken together, this study reveals a new mechanism where a SCFSNIPER7 complex regulates CDC48 unfoldase levels and modulates immune output.
Assuntos
Proteínas F-Box , Imunidade Vegetal , Proteômica , Proteínas F-Box/metabolismo , Proteólise , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
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.
Assuntos
Ascomicetos/metabolismo , Mitocôndrias/metabolismo , Fitoquelatinas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Catálise , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologiaRESUMO
Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.
Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Ascomicetos/fisiologia , Resistência à Doença , Glucosinolatos/biossíntese , Doenças das Plantas/microbiologia , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Indóis , Epiderme Vegetal/citologia , Proteínas Recombinantes/metabolismoRESUMO
Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.
Assuntos
Arabidopsis/microbiologia , Basidiomycota/fisiologia , Celulose/metabolismo , Exorribonucleases/metabolismo , Simbiose/fisiologia , Trioses/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Exorribonucleases/genética , Regulação da Expressão Gênica de Plantas , Mutação , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Plântula/metabolismo , Plântula/microbiologia , Transdução de SinaisRESUMO
The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Interações Hospedeiro-Patógeno , Mitocôndrias/metabolismo , N-Glicosil Hidrolases/metabolismo , Epiderme Vegetal/metabolismo , Folhas de Planta/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Resistência à Doença , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , Oxirredução , Peroxissomos/metabolismo , Doenças das Plantas/microbiologia , Multimerização Proteica , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
Stringent modulation of immune signaling in plants is necessary to enable a rapid response to pathogen attack without spurious defense activation. To identify genes involved in plant immunity, a forward genetic screen for enhancers of the autoimmune snc1 (suppressor of npr1, constitutive 1) mutant was conducted. The snc1 mutant contains a gain-of-function mutation in a gene encoding a NOD-like receptor (NLR) protein. The isolated muse7 (mutant, snc1-enhancing, 7) mutant was shown to confer a reversion to autoimmune phenotypes in the wild-type-like mos4 (modifier of snc1, 4) snc1 background. Positional cloning revealed that MUSE7 encodes an evolutionarily conserved putative kinase substrate of unknown function. The muse7 single mutants display enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. While transcription of SNC1 is not enhanced, elevated SNC1 protein accumulation is associated with mutations in muse7. Accumulation of two additional NLR proteins, RPS2 (RESISTANCE TO PSEUDOMONAS SYRINGAE 2) and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE pv. MACULICOLA 1), was also observed in muse7 plants. Although proteasome-mediated degradation of NLR proteins is a well studied event in plant immunity, no interactions were detected between MUSE7 and selected components of this pathway. This study has demonstrated a role for MUSE7 in modulating plant immune responses through negatively affecting NLR accumulation, and will benefit future studies of MUSE7 homologs in other species.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas NLR/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Mutação , Proteínas NLR/genética , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Plantas Geneticamente Modificadas/genéticaRESUMO
To detect potential pathogens, plants perceive the fungal polysaccharide chitin through receptor complexes containing lysin motif receptor-like kinases (LysM-RLKs). To investigate the ligand-induced spatial dynamics of chitin receptor components, we studied the subcellular behaviour of two Arabidopsis thaliana LysM-RLKs involved in chitin signalling, CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) and LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5. We performed standard and quantitative confocal laser scanning microscopy on stably transformed A. thaliana plants expressing fluorescently tagged CERK1 and LYK5 from their native promoters. Microscopy approaches were complemented by biochemical analyses in plants and in vitro. Both CERK1 and LYK5 localized to the plasma membrane and showed constitutive endomembrane trafficking. After chitin treatment, however, CERK1 remained at the plasma membrane while LYK5 relocalized into mobile intracellular vesicles. Detailed analyses revealed that chitin perception transiently induced the internalization of LYK5 into late endocytic compartments. Plants that lacked CERK1 or expressed an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. CERK1 could phosphorylate LYK5 in vitro and chitin treatment induced CERK1-dependent phosphorylation of LYK5 in planta. Our results suggest that chitin-induced phosphorylation by CERK1 triggers LYK5 internalization. Thus, our work identifies phosphorylation as a key regulatory step in endocytosis of plant RLKs and also provides evidence for receptor complex dissociation after ligand perception.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Quitina/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Arabidopsis/citologia , Endocitose , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Complexos Ubiquitina-Proteína Ligase/fisiologia , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Resistência à Doença/genética , Imunidade Vegetal , Proteólise , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinaçãoRESUMO
Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.
Assuntos
Ascomicetos/fisiologia , Genes de Plantas , Glucosiltransferases/genética , Hordeum/genética , Hordeum/microbiologia , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/microbiologia , Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Glucosiltransferases/metabolismo , Hordeum/enzimologia , Epiderme Vegetal/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Análise de Sequência de DNARESUMO
Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.
Assuntos
Alelos , Arabidopsis/genética , Aptidão Genética/genética , Variação Genética/genética , Anquirinas/genética , Anquirinas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudo de Associação Genômica Ampla , Dados de Sequência Molecular , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/parasitologia , Locos de Características QuantitativasRESUMO
Chitin acts as a pathogen-associated molecular pattern from fungal pathogens whose perception triggers a range of defense responses. We show that LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2), the Arabidopsis homolog of a rice chitin receptor-like protein, mediates a reduction in molecular flux via plasmodesmata in the presence of chitin. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not to the flagellin derivative flg22. Surprisingly, the chitin-recognition receptor CHITIN ELCITOR RECEPTOR KINASE 1 (CERK1) is not required for chitin-induced changes to plasmodesmata flux, suggesting that there are at least two chitin-activated response pathways in Arabidopsis and that LYM2 is not required for CERK1-mediated chitin-triggered defense responses, indicating that these pathways are independent. In accordance with a role in the regulation of intercellular flux, LYM2 is resident at the plasma membrane and is enriched at plasmodesmata. Chitin-triggered regulation of molecular flux between cells is required for defense responses against the fungal pathogen Botrytis cinerea, and thus we conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Botrytis , Comunicação Celular/imunologia , Quitina/metabolismo , Doenças das Plantas/imunologia , Plasmodesmos/metabolismo , Receptores de Superfície Celular/metabolismo , Compostos de Anilina , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia Confocal , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/fisiologia , Azul TripanoRESUMO
The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Brassica napus/fisiologia , Doenças das Plantas/microbiologia , Verticillium/fisiologia , Xilema/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Brassica napus/citologia , Brassica napus/genética , Brassica napus/microbiologia , Diferenciação Celular , Secas , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Especificidade de Órgãos , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Feixe Vascular de Plantas/citologia , Feixe Vascular de Plantas/genética , Feixe Vascular de Plantas/microbiologia , Feixe Vascular de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Água/metabolismo , Xilema/citologia , Xilema/genética , Xilema/microbiologiaRESUMO
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.
Assuntos
Ascomicetos/patogenicidade , Proteínas de Plantas/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Ascomicetos/classificação , Ascomicetos/fisiologia , Filogenia , Plantas Geneticamente Modificadas , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Non-host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre- and post-invasive resistance responses. Pre-invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non-host resistance to P. infestans, a genetic screen was performed by re-mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2-1 erp1-3 and pen2-1 erp1-4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T-DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen-inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non-autonomous defense responses against invasive filamentous pathogens.
Assuntos
Arabidopsis/microbiologia , Ésteres/metabolismo , Phytophthora infestans/patogenicidade , Esteróis/biossíntese , Arabidopsis/genética , Genes de Plantas , Mutação , Proteínas de Plantas/metabolismo , Frações Subcelulares/metabolismoRESUMO
The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane-localized receptor complexes. A critical step in their activation is ligand-induced homo- or hetero-oligomerization of leucine-rich repeat (LRR)- and/or lysin motif (LysM) receptor-like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen-associated molecular patterns (PAMPs), including the bacterial flagellin-derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont-derived (lipo)-chitooligosaccharides. The structurally related chitin-oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM-RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin-like domain (MLD)-LRR-RLK Symbiosis Receptor-like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR- and LysM-mediated signalling, the involvement of MLD-LRR-RLKs in symbiosis and defence, and the role of endocytosis in RLK function.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Plantas/microbiologia , Proteínas Quinases/metabolismo , Simbiose/fisiologia , Motivos de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Quitina/metabolismo , Endocitose , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , RhizobiumRESUMO
Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens.