RESUMEN
Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Phytophthora infestans/fisiología , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Mapas de Interacción de Proteínas , Proteínas SNARE/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
Xylella fastidiosa is the causal agent of important crop diseases and is transmitted by xylem-sap-feeding insects. The bacterium colonizes xylem vessels and can persist with a commensal or pathogen lifestyle in more than 500 plant species. In the past decade, reports of X. fastidiosa across the globe have dramatically increased its known occurrence. This raises important questions: How does X. fastidiosa interact with the different host plants? How does the bacterium interact with the plant immune system? How does it influence the host's microbiome? We discuss recent strain genetic typing and plant transcriptome and microbiome analyses, which have advanced our understanding of factors that are important for X. fastidiosa plant infection.
Asunto(s)
Microbiota , Xylella , Enfermedades de las Plantas/microbiología , PlantasRESUMEN
Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high-throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non-redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image-based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.
Asunto(s)
Membrana Celular/metabolismo , Estomas de Plantas/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endosomas/metabolismo , Presión Osmótica , Estomas de Plantas/citología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Proteínas SNARE/genética , Análisis de la Célula Individual , Proteínas de Unión al GTP rab/genéticaRESUMEN
Plants detect and respond to pathogen invasion with membrane-localized pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and activate downstream immune responses. Here we report that Arabidopsis thaliana LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1), a coreceptor of the receptor-like kinase FERONIA, regulates PRR signaling. In a forward genetic screen for suppressors of enhanced disease resistance 1 (edr1), we identified the point mutation llg1-3, which suppresses edr1 disease resistance but does not affect plant growth and development. The llg1 mutants show enhanced susceptibility to various virulent pathogens, indicating that LLG1 has an important role in plant immunity. LLG1 constitutively associates with the PAMP receptor FLAGELLIN SENSING 2 (FLS2) and the elongation factor-Tu receptor, and forms a complex with BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 in a ligand-dependent manner, indicating that LLG1 functions as a key component of PAMP-recognition immune complexes. Moreover, LLG1 contributes to accumulation and ligand-induced degradation of FLS2, and is required for downstream innate immunity responses, including ligand-induced phosphorylation of BOTRYTIS-INDUCED KINASE 1 and production of reactive oxygen species. Taken together, our findings reveal that LLG1 associates with PAMP receptors and modulates their function to regulate disease responses. As LLG1 functions as a coreceptor of FERONIA and plays central roles in plant growth and development, our findings indicate that LLG1 participates in separate pathways, and may suggest a potential connection between development and innate immunity in plants.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Inmunidad de la Planta , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Ligadas a GPI/genética , Inmunidad Innata/genética , Mutación , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosfotransferasas/inmunología , Fosfotransferasas/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismoRESUMEN
High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule-targeted herbicides and demonstrate that high throughput confocal imaging with integrated image analysis by CellArchitect can distinguish effects induced by the known herbicides indaziflam and trifluralin. The same platform was used to examine 6 other compounds with herbicidal activity, and at least 3 different effects induced by these compounds were profiled. We further show that CellArchitect can detect subcellular patterns tagged by actin and endoplasmic reticulum markers. Thus, the platform developed here can be used to automate image analysis of complex subcellular patterns for purposes such as herbicide discovery and mode of action characterisation. The capacity to use this tool to quantitatively characterize cellular responses lends itself to application across many areas of biology.
Asunto(s)
Herbicidas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Microtúbulos/efectos de los fármacos , Imagen Óptica/métodos , Moduladores de Tubulina/farmacología , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Indenos/farmacología , Microtúbulos/metabolismo , Unión Proteica , Triazinas/farmacología , Trifluralina/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
Sensing of potential pathogenic bacteria is of critical importance for immunity. In plants, this involves plasma membrane-resident pattern recognition receptors, one of which is the FLAGELLIN SENSING 2 (FLS2) receptor kinase. Ligand-activated FLS2 receptors are internalized into endosomes. However, the extent to which these spatiotemporal dynamics are generally present among pattern recognition receptors (PRRs) and their regulation remain elusive. Using live-cell imaging, we show that at least three other receptor kinases associated with plant immunity, PEP RECEPTOR 1/2 (PEPR1/2) and EF-TU RECEPTOR (EFR), internalize in a ligand-specific manner. In all cases, endocytosis requires the coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1), and thus depends on receptor activation status. We also show the internalization of liganded FLS2, suggesting the transport of signaling competent receptors. Trafficking of activated PRRs requires clathrin and converges onto the same endosomal vesicles that are also shared with the hormone receptor BRASSINOSTERIOD INSENSITIVE 1 (BRI1). Importantly, clathrin-dependent endocytosis participates in plant defense against bacterial infection involving FLS2-mediated stomatal closure and callose deposition, but is uncoupled from activation of the flagellin-induced oxidative burst and MAP kinase signaling. In conclusion, immunity mediated by pattern recognition receptors depends on clathrin, a critical component for the endocytosis of signaling competent receptors into a common endosomal pathway.
Asunto(s)
Arabidopsis/inmunología , Clatrina/metabolismo , Endocitosis , Nicotiana/inmunología , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Autofagia , Endosomas/metabolismo , Flagelina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ligandos , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Estomas de Plantas/fisiología , Transducción de Señal , Nicotiana/metabolismoRESUMEN
Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening.
Asunto(s)
Arabidopsis/genética , Pared Celular/metabolismo , Simulación por Computador , Vicia faba/genética , Arabidopsis/fisiología , Fenómenos Biomecánicos , Estomas de Plantas/genética , Estomas de Plantas/fisiología , Vicia faba/fisiologíaRESUMEN
Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Fosfoproteínas Fosfatasas/inmunología , Inmunidad de la Planta/fisiología , Proteínas Serina-Treonina Quinasas/inmunología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , Fosfoproteínas Fosfatasas/genética , Fosforilación/genética , Fosforilación/inmunología , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H(+)-ATPase activity. The plasma membrane H(+)-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas Portadoras/metabolismo , Membrana Celular/enzimología , Inmunidad de la Planta/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Aminoácidos/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/química , Proteínas Portadoras/química , Membrana Celular/efectos de los fármacos , Flagelina/farmacología , Técnicas de Inactivación de Genes , Indenos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Datos de Secuencia Molecular , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Enfermedades de las Plantas/microbiología , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/fisiología , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Flagelina/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Inmunidad de la Planta , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Estrés Oxidativo/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Ascomicetos/inmunología , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas Serina-Treonina Quinasas/genética , Pseudomonas syringae/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Xantina Oxidasa/metabolismoRESUMEN
Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
Asunto(s)
Fosfoinositido Fosfolipasa C/genética , Inmunidad de la Planta/genética , Solanum lycopersicum/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/enzimología , Familia de Multigenes , Fosfoinositido Fosfolipasa C/biosíntesis , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Proteínas Quinasas/genéticaRESUMEN
The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endosomas/metabolismo , Proteómica/métodos , Vías Secretoras , Cromatografía de Afinidad , Endocitosis , Proteínas Fluorescentes Verdes/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Fracciones Subcelulares/metabolismo , Red trans-Golgi/metabolismoAsunto(s)
Arabidopsis , Citrus sinensis , Citrus , Xanthomonas , Xylella , Arabidopsis/genética , Enfermedades de las PlantasRESUMEN
The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Oomicetos/fisiología , Enfermedades de las Plantas/inmunología , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Interacciones Huésped-Patógeno , Péptidos y Proteínas de Señalización Intracelular , Mutación , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Plasmodesmos/metabolismoRESUMEN
The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Endocitosis , Proteínas Fúngicas/metabolismo , Inmunidad de la Planta , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Membrana Celular/metabolismo , Cladosporium , Endosomas/metabolismo , Ligandos , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Modelos Biológicos , Plantas Modificadas Genéticamente , Unión Proteica , Nicotiana/genéticaRESUMEN
Plant NB-LRR proteins confer resistance to multiple pathogens, including viruses. Although the recognition of viruses by NB-LRR proteins is highly specific, previous studies have suggested that NB-LRR activation results in a response that targets all viruses in the infected cell. Using an inducible system to activate NB-LRR defenses, we find that NB-LRR signaling does not result in the degradation of viral transcripts, but rather prevents them from associating with ribosomes and translating their genetic material. This indicates that defense against viruses involves the repression of viral RNA translation. This repression is specific to viral transcripts and does not involve a global shutdown of host cell translation. As a consequence of the repression of viral RNA translation, NB-LRR responses induce a dramatic increase in the biogenesis of RNA processing bodies (PBs). We demonstrate that other pathways that induce translational repression, such as UV irradiation and RNAi, also induce PBs. However, by investigating the phosphorylation status of eIF2α and by using suppressors of RNAi we show that the mechanisms leading to PB induction by NB-LRR signaling are different from these stimuli, thus defining a distinct type of translational control and anti-viral mechanism in plants.
Asunto(s)
Proteínas NLR/metabolismo , Biosíntesis de Proteínas/efectos de la radiación , Interferencia de ARN/efectos de la radiación , Procesamiento Postranscripcional del ARN/efectos de la radiación , ARN Viral/genética , Transducción de Señal , Estrés Fisiológico/efectos de la radiación , Rayos Ultravioleta , Hojas de la Planta/genética , Hojas de la Planta/efectos de la radiación , Potexvirus/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Reproducibilidad de los Resultados , Nicotiana/genéticaRESUMEN
Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink-source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Plasmodesmos/ultraestructura , Algoritmos , Arabidopsis/efectos de los fármacos , Transporte Biológico , Comunicación Celular/fisiología , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Citocinesis/efectos de los fármacos , Proteínas Fluorescentes Verdes , Manitol/farmacología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Epidermis de la Planta/efectos de los fármacos , Epidermis de la Planta/crecimiento & desarrollo , Epidermis de la Planta/ultraestructura , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/ultraestructura , Plantas Modificadas Genéticamente , Plasmodesmos/efectos de los fármacos , Ácido Salicílico/farmacologíaRESUMEN
The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity.
Asunto(s)
Proteínas de Arabidopsis/genética , Endocitosis/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Inmunidad de la Planta/genética , Proteínas Quinasas/genética , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/genética , Endosomas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Unión al GTP rab5/genéticaRESUMEN
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.