RESUMEN
Quantitative disease resistance (QDR) represents the predominant form of resistance in natural populations and crops. Surprisingly, very limited information exists on the biomolecular network of the signaling machineries underlying this form of plant immunity. This lack of information may result from its complex and quantitative nature. Here, we used an integrative approach including genomics, network reconstruction, and mutational analysis to identify and validate molecular networks that control QDR in Arabidopsis thaliana in response to the bacterial pathogen Xanthomonas campestris To tackle this challenge, we first performed a transcriptomic analysis focused on the early stages of infection and using transgenic lines deregulated for the expression of RKS1, a gene underlying a QTL conferring quantitative and broad-spectrum resistance to XcampestrisRKS1-dependent gene expression was shown to involve multiple cellular activities (signaling, transport, and metabolism processes), mainly distinct from effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses already characterized in Athaliana Protein-protein interaction network reconstitution then revealed a highly interconnected and distributed RKS1-dependent network, organized in five gene modules. Finally, knockout mutants for 41 genes belonging to the different functional modules of the network revealed that 76% of the genes and all gene modules participate partially in RKS1-mediated resistance. However, these functional modules exhibit differential robustness to genetic mutations, indicating that, within the decentralized structure of the QDR network, some modules are more resilient than others. In conclusion, our work sheds light on the complexity of QDR and provides comprehensive understanding of a QDR immune network.
Asunto(s)
Resistencia a la Enfermedad/inmunología , Susceptibilidad a Enfermedades/inmunología , Interacciones Huésped-Patógeno , Inmunomodulación , Modelos Biológicos , Enfermedades de las Plantas/etiología , Inmunidad de la Planta , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Fenotipo , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , TranscriptomaRESUMEN
BACKGROUND: The ongoing adaptation of plants to their environment is the basis for their survival. In this adaptation, mechanoperception of gravity and local curvature plays a role of prime importance in finely regulating growth and ensuring a dynamic balance preventing buckling. However, the abiotic environment is not the exclusive cause of mechanical stimuli. Biotic interactions between plants and microorganisms also involve physical forces and potentially mechanoperception. Whether pathogens trigger mechanoperception in plants and the impact of mechanotransduction on the regulation of plant defense remains however elusive. RESULTS: Here, we found that the perception of pathogen-derived mechanical cues by microtubules potentiates the spatio-temporal implementation of plant immunity to fungus. By combining biomechanics modeling and image analysis of the post-invasion stage, we reveal that fungal colonization releases plant cell wall-born tension locally, causing fluctuations of tensile stress in walls of healthy cells distant from the infection site. In healthy cells, the pathogen-derived mechanical cues guide the reorganization of mechanosensing cortical microtubules (CMT). The anisotropic patterning of CMTs is required for the regulation of immunity-related genes in distal cells. The CMT-mediated mechanotransduction of pathogen-derived cues increases Arabidopsis disease resistance by 40% when challenged with the fungus Sclerotinia sclerotiorum. CONCLUSIONS: CMT anisotropic patterning triggered by pathogen-derived mechanical cues activates the implementation of early plant defense in cells distant from the infection site. We propose that the mechano-signaling triggered immunity (MTI) complements the molecular signals involved in pattern and effector-triggered immunity.
Asunto(s)
Arabidopsis , Mecanotransducción Celular , Señales (Psicología) , Plantas , Transducción de Señal , Inmunidad de la Planta , Arabidopsis/genética , Enfermedades de las Plantas , Regulación de la Expresión Génica de las PlantasRESUMEN
The broad host range necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen of many oil and vegetable crops. Plant genes conferring complete resistance against S. sclerotiorum have not been reported. Instead, plant populations challenged by S. sclerotiorum exhibit a continuum of partial resistance designated as quantitative disease resistance (QDR). Because of their complex interplay and their small phenotypic effect, the functional characterization of QDR genes remains limited. How broad host range necrotrophic fungi manipulate plant programmed cell death is for instance largely unknown. Here, we designed a time-resolved automated disease phenotyping pipeline enabling high-throughput disease lesion measurement with high resolution, low footprint at low cost. We could accurately recover contrasted disease responses in several pathosystems using this system. We used our phenotyping pipeline to assess the kinetics of disease symptoms caused by seven S. sclerotiorum isolates on six A. thaliana natural accessions with unprecedented resolution. Large effect polymorphisms common to the most resistant A. thaliana accessions identified highly divergent alleles of the nucleotide-binding site leucine-rich repeat gene LAZ5 in the resistant accessions Rubezhnoe and Lip-0. We show that impaired LAZ5 expression in laz5.1 mutant lines and in A. thaliana Rub natural accession correlate with enhanced QDR to S. sclerotiorum. These findings illustrate the value of time-resolved image-based phenotyping for unravelling the genetic bases of complex traits such as QDR. Our results suggest that S. sclerotiorum manipulates plant sphingolipid pathways guarded by LAZ5 to trigger programmed cell death and cause disease.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ascomicetos , Genes de Plantas/fisiología , Proteínas NLR/genética , Enfermedades de las Plantas/microbiología , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/fisiología , Resistencia a la Enfermedad/genética , Genes de Plantas/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad/genética , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas NLR/fisiología , Enfermedades de las Plantas/inmunología , Carácter Cuantitativo HeredableRESUMEN
Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/fisiología , Terpenos/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas Argonautas/química , Biocatálisis , Membrana Celular/metabolismo , Silenciador del Gen , Proteínas de la Membrana/química , MicroARNs/genética , Datos de Secuencia Molecular , Esteroles/biosíntesisRESUMEN
BACKGROUND: Several regulators of programmed cell death (PCD) in plants encode proteins with putative lipid-binding domains. Among them, VAD1 is a regulator of PCD propagation harboring a GRAM putative lipid-binding domain. However the function of VAD1 at the subcellular level is unknown and the domain architecture of VAD1 has not been analyzed in details. RESULTS: We analyzed sequence conservation across the plant kingdom in the VAD1 protein and identified an uncharacterized VASt (VAD1 Analog of StAR-related lipid transfer) domain. Using profile hidden Markov models (profile HMMs) and phylogenetic analysis we found that this domain is conserved among eukaryotes and generally associates with various lipid-binding domains. Proteins containing both a GRAM and a VASt domain include notably the yeast Ysp2 cell death regulator and numerous uncharacterized proteins. Using structure-based phylogeny, we found that the VASt domain is structurally related to Bet v1-like domains. CONCLUSION: We identified a novel protein domain ubiquitous in Eukaryotic genomes and belonging to the Bet v1-like superfamily. Our findings open perspectives for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.
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Eucariontes/química , Lípidos/química , Filogenia , Proteínas/química , Secuencia de Aminoácidos , Animales , Secuencia Conservada/genética , Eucariontes/genética , Eucariontes/metabolismo , Genoma , Humanos , Metabolismo de los Lípidos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Snf1-related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA-independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site-directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two-dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA-responsiveness.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Biocatálisis/efectos de los fármacos , Western Blotting , Electroforesis en Gel Bidimensional , Activación Enzimática/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Concentración Osmolar , Fosforilación , Reguladores del Crecimiento de las Plantas/farmacología , Unión Proteica , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Pathogen infection triggers extensive reprogramming of the plant transcriptome, including numerous genes the function of which is unknown. Due to their wide taxonomic distribution, genes encoding proteins with Domains of Unknown Function (DUFs) activated upon pathogen challenge likely play important roles in disease. In Arabidopsis thaliana, we identified thirteen genes harboring a DUF4228 domain in the top 10% most induced genes after infection by the fungal pathogen Sclerotinia sclerotiorum. Based on functional information collected through homology and contextual searches, we propose to refer to this domain as the pathogen and abiotic stress response, cadmium tolerance, disordered region-containing (PADRE) domain. Genome-wide and phylogenetic analyses indicated that PADRE is specific to plants and diversified into 10 subfamilies early in the evolution of Angiosperms. PADRE typically occurs in small single-domain proteins with a bipartite architecture. PADRE N-terminus harbors conserved sequence motifs, while its C-terminus includes an intrinsically disordered region with multiple phosphorylation sites. A pangenomic survey of PADRE genes expression upon S. sclerotiorum inoculation in Arabidopsis, castor bean, and tomato indicated consistent expression across species within phylogenetic groups. Multi-stress expression profiling and co-expression network analyses associated AtPADRE genes with the induction of anthocyanin biosynthesis and responses to chitin and to hypoxia. Our analyses reveal patterns of sequence and expression diversification consistent with the evolution of a role in disease resistance for an uncharacterized family of plant genes. These findings highlight PADRE genes as prime candidates for the functional dissection of mechanisms underlying plant disease resistance to fungi.
RESUMEN
Progress in remote sensing and robotic technologies decreases the hardware costs of phenotyping. Here, we first review cost-effective imaging devices and environmental sensors, and present a trade-off between investment and manpower costs. We then discuss the structure of costs in various real-world scenarios. Hand-held low-cost sensors are suitable for quick and infrequent plant diagnostic measurements. In experiments for genetic or agronomic analyses, (i) major costs arise from plant handling and manpower; (ii) the total costs per plant/microplot are similar in robotized platform or field experiments with drones, hand-held or robotized ground vehicles; (iii) the cost of vehicles carrying sensors represents only 5-26% of the total costs. These conclusions depend on the context, in particular for labor cost, the quantitative demand of phenotyping and the number of days available for phenotypic measurements due to climatic constraints. Data analysis represents 10-20% of total cost if pipelines have already been developed. A trade-off exists between the initial high cost of pipeline development and labor cost of manual operations. Overall, depending on the context and objsectives, "cost-effective" phenotyping may involve either low investment ("affordable phenotyping"), or initial high investments in sensors, vehicles and pipelines that result in higher quality and lower operational costs.
Asunto(s)
Análisis Costo-Beneficio/métodos , Plantas/genética , Sistemas de Información , FenotipoRESUMEN
Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid-binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant vad1-1 has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, associated with defense gene expression and increased resistance to Pseudomonas strains. To test the potential of ectopic expression of VAD1 to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure-function analysis of VAD1 by transient over-expression in Nicotiana benthamiana and by complementation of the mutant vad1-1. We found that (i) overexpression of VAD1 controls negatively the HR cell death and defense expression either transiently in Nicotiana benthamania or in Arabidopsis plants in response to avirulent strains of Pseudomonas syringae, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/inmunología , Muerte Celular/inmunología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/inmunología , Muerte Celular/genética , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Núcleo Celular/microbiología , Citosol/inmunología , Citosol/metabolismo , Citosol/microbiología , Prueba de Complementación Genética , Mutación , Células Vegetales/inmunología , Células Vegetales/metabolismo , Células Vegetales/microbiología , Enfermedades de las Plantas/genética , Dominios Proteicos , Pseudomonas syringae/crecimiento & desarrollo , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
NADPH-dependent thioredoxin reductases (NTR) are homodimeric enzymes that reduce thioredoxins. Two genes encoding NADPH-dependent thioredoxin reductases (AtNTRA and AtNTRB) were found in the genome of Arabidopsis thaliana. These originated from a recent duplication event and the encoded proteins are highly homologous. Previously, AtNTRA was shown to encode a dual targeted cytosol and mitochondrial protein. Here, we show that the AtNTRB gene encodes two mRNAs, presumably by initiating transcription at two different sites. The longer mRNA encodes a precursor polypeptide that is actively imported into mitochondria by a cleavage-associated mechanism, while the shorter mRNA encodes a cytosolic isoform. Isolation of Arabidopsis mutants with knocked-out AtNTRA or AtNTRB genes allowed us to prove that both genes encode cytosolic and mitochondrial isoforms. Interestingly, AtNTRB appeared to express the major mitochondrial NTR, while AtNTRA expresses as the major cytosolic isoform, suggesting that these two recently duplicated genes are evolving towards a specific function.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Mitocondrias/enzimología , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Citosol/química , Citosol/enzimología , Duplicación de Gen , Mitocondrias/química , Mitocondrias/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Alineación de Secuencia , Reductasa de Tiorredoxina-Disulfuro/análisis , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Transcripción GenéticaRESUMEN
One of the most efficient plant resistance reactions to pathogen attack is the hypersensitive response, a form of programmed cell death at infection sites. The Arabidopsis transcription factor MYB30 is a positive regulator of hypersensitive cell death responses. Here we show that MIEL1 (MYB30-Interacting E3 Ligase1), an Arabidopsis RING-type E3 ubiquitin ligase that interacts with and ubiquitinates MYB30, leads to MYB30 proteasomal degradation and downregulation of its transcriptional activity. In non-infected plants, MIEL1 attenuates cell death and defence through degradation of MYB30. Following bacterial inoculation, repression of MIEL1 expression removes this negative regulation allowing sufficient MYB30 accumulation in the inoculated zone to trigger the hypersensitive response and restrict pathogen growth. Our work underlines the important role played by ubiquitination to control the hypersensitive response and highlights the sophisticated fine-tuning of plant responses to pathogen attack. Overall, this work emphasizes the importance of protein modification by ubiquitination during the regulation of transcriptional responses to stress in eukaryotic cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteolisis , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica de las Plantas , Microscopía Confocal , Células Vegetales/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transporte de Proteínas , Pseudomonas syringae/fisiología , Nicotiana/genética , Factores de Transcripción/genética , Transcripción Genética , UbiquitinaciónRESUMEN
NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Glutatión/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Antocianinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diploidia , Activación Enzimática , Fertilidad , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Glutarredoxinas , Modelos Biológicos , Mutación/genética , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas/metabolismo , Fenotipo , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Polen/metabolismo , Plantones/metabolismo , Semillas/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genéticaRESUMEN
An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.