RESUMEN
The endoplasmic reticulum (ER) is the entry point to the secretory pathway and, as such, is critical for adaptive responses to biotic stress, when the demand for de novo synthesis of immunity-related proteins and signalling components increases significantly. Successful phytopathogens have evolved an arsenal of small effector proteins which collectively reconfigure multiple host components and signalling pathways to promote virulence; a small, but important, subset of which are targeted to the endomembrane system including the ER. We identified and validated a conserved C-terminal tail-anchor motif in a set of pathogen effectors known to localize to the ER from the oomycetes Hyaloperonospora arabidopsidis and Plasmopara halstedii (downy mildew of Arabidopsis and sunflower, respectively) and used this protein topology to develop a bioinformatic pipeline to identify putative ER-localized effectors within the effectorome of the related oomycete, Phytophthora infestans, the causal agent of potato late blight. Many of the identified P. infestans tail-anchor effectors converged on ER-localized NAC transcription factors, indicating that this family is a critical host target for multiple pathogens.
Asunto(s)
Oomicetos , Phytophthora infestans , Plantas/metabolismo , Factores de Transcripción/metabolismo , Virulencia , Retículo Endoplásmico/metabolismo , Enfermedades de las PlantasRESUMEN
The host range of parasites is an important factor in assessing the dynamics of disease epidemics. The evolution of pathogens to accommodate new hosts may lead to host range expansion, a process the molecular bases of which are largely enigmatic. The fungus Sclerotinia sclerotiorum has been reported to parasitize more than 400 plant species from diverse eudicot families while its close relative, S. trifoliorum, is restricted to plants from the Fabaceae family. We analyzed S. sclerotiorum global transcriptome reprogramming on hosts from six botanical families and reveal a flexible, host-specific transcriptional program. We generated a chromosome-level genome assembly for S. trifoliorum and found near-complete gene space conservation in two representative strains of broad and narrow host range Sclerotinia species. However, S. trifoliorum showed increased sensitivity to the Brassicaceae defense compound camalexin. Comparative analyses revealed a lack of transcriptional response to camalexin in the S. trifoliorum strain and suggest that regulatory variation in detoxification and effector genes at the population level may associate with the genetic accommodation of Brassicaceae in the Sclerotinia host range. Our work proposes transcriptional plasticity and the co-existence of signatures for generalist and polyspecialist adaptive strategies in the genome of a plant pathogen.
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Señales (Psicología) , Especificidad del Huésped , Humanos , Enfermedades de las Plantas/microbiología , Plantas/microbiología , TranscriptomaRESUMEN
Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.
RESUMEN
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
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
Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad-spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad-spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern-triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad-spectrum resistant lines. HR triggered by PhRXLRC01 co-segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad-spectrum resistance gene identification in complex crop genomes such as sunflower.
Asunto(s)
Helianthus/metabolismo , Helianthus/microbiología , Oomicetos/patogenicidad , Enfermedades de las Plantas/microbiología , Mapeo Cromosómico , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/fisiología , Genotipo , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated Pl23-Pl32 according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower.
RESUMEN
The obligate biotroph oomycete Plasmopara halstedii causes downy mildew on sunflower crop, Helianthus annuus. The breakdown of several Pl resistance genes used in sunflower hybrids over the last 25 years came along with the appearance of new Pl. halstedii isolates showing modified virulence profiles. In oomycetes, two classes of effector proteins, key players of pathogen virulence, are translocated into the host: RXLR and CRN effectors. We identified 54 putative CRN or RXLR effector genes from transcriptomic data and analyzed their genetic diversity in seven Pl. halstedii pathotypes representative of the species variability. Pl. halstedii effector genes were on average more polymorphic at both the nucleic and protein levels than random non-effector genes, suggesting a potential adaptive dynamics of pathogen virulence over the last 25 years. Twenty-two KASP (Competitive Allele Specific PCR) markers designed on polymorphic effector genes were genotyped on 35 isolates belonging to 14 Pl. halstedii pathotypes. Polymorphism analysis based on eight KASP markers aims at proposing a determination key suitable to classify the eight multi-isolate pathotypes into six groups. This is the first report of a molecular marker set able to discriminate Pl. halstedii pathotypes based on the polymorphism of pathogenicity effectors. Compared to phenotypic tests handling living spores used until now to discriminate Pl. halstedii pathotypes, this set of molecular markers constitutes a first step in faster pathotype diagnosis of Pl. halstedii isolates. Hence, emerging sunflower downy mildew isolates could be more rapidly characterized and thus, assessment of plant resistance breakdown under field conditions should be improved.
Asunto(s)
Helianthus/microbiología , Oomicetos/genética , Polimorfismo Genético , Alelos , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Oomicetos/clasificación , Oomicetos/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Polimorfismo de Nucleótido Simple , Virulencia/genéticaRESUMEN
Plasmopara halstedii is an obligate biotrophic oomycete causing downy mildew disease on sunflower, Helianthus annuus, an economically important oil crop. Severe symptoms of the disease (e.g., plant dwarfism, leaf bleaching, sporulation and production of infertile flower) strongly impair seed yield. Pl resistance genes conferring resistance to specific P. halstedii pathotypes were located on sunflower genetic map but yet not cloned. They are present in cultivated lines to protect them against downy mildew disease. Among the 16 different P. halstedii pathotypes recorded in France, pathotype 710 is frequently found, and therefore continuously controlled in sunflower by different Pl genes. High-throughput sequencing of cDNA from P. halstedii led us to identify potential effectors with the characteristic RXLR or CRN motifs described in other oomycetes. Expression of six P. halstedii putative effectors, five RXLR and one CRN, was analyzed by qRT-PCR in pathogen spores and in the pathogen infecting sunflower leaves and selected for functional analyses. We developed a new method for transient expression in sunflower plant leaves and showed for the first time subcellular localization of P. halstedii effectors fused to a fluorescent protein in sunflower leaf cells. Overexpression of the CRN and of 3 RXLR effectors induced hypersensitive-like cell death reactions in some sunflower near-isogenic lines resistant to pathotype 710 and not in susceptible corresponding lines, suggesting they could be involved in Pl loci-mediated resistances.
RESUMEN
Downy mildew of sunflower is caused by Plasmopara halstedii (Farlow) Berlese & de Toni. Plasmopara halstedii is an obligate biotrophic oomycete pathogen that attacks annual Helianthus species and cultivated sunflower, Helianthus annuus. Depending on the sunflower developmental stage at which infection occurs, the characteristic symptoms range from young seedling death, plant dwarfing, leaf bleaching and sporulation to the production of infertile flowers. Downy mildew attacks can have a great economic impact on sunflower crops, and several Pl resistance genes are present in cultivars to protect them against the disease. Nevertheless, some of these resistances have been overcome by the occurrence of novel isolates of the pathogen showing increased virulence. A better characterization of P. halstedii infection and dissemination mechanisms, and the identification of the molecular basis of the interaction with sunflower, is a prerequisite to efficiently fight this pathogen. This review summarizes what is currently known about P. halstedii, provides new insights into its infection cycle on resistant and susceptible sunflower lines using scanning electron and light microscopy imaging, and sheds light on the pathogenicity factors of P. halstedii obtained from recent molecular data. TAXONOMY: Kingdom Stramenopila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Plasmopara; Species Plasmopara halstedii. DISEASE SYMPTOMS: Sunflower seedling damping off, dwarfing of the plant, bleaching of leaves, starting from veins, and visible white sporulation, initially on the lower side of cotyledons and leaves. Plasmopara halstedii infection may severely impact sunflower seed yield. INFECTION PROCESS: In spring, germination of overwintered sexual oospores leads to sunflower root infection. Intercellular hyphae are responsible for systemic plant colonization and the induction of disease symptoms. Under humid and fresh conditions, dissemination structures are produced by the pathogen on all plant organs to release asexual zoosporangia. These zoosporangia play an important role in pathogen dissemination, as they release motile zoospores that are responsible for leaf infections on neighbouring plants. DISEASE CONTROL: Disease control is obtained by both chemical seed treatment (mefenoxam) and the deployment of dominant major resistance genes, denoted Pl. However, the pathogen has developed fungicide resistance and has overcome some plant resistance genes. Research for more sustainable strategies based on the identification of the molecular basis of the interaction are in progress. USEFUL WEBSITES: http://www.heliagene.org/HP, http://lipm-helianthus.toulouse.inra.fr/dokuwiki/doku.php?id=start, https://www.heliagene.org/PlasmoparaSpecies (soon available).
Asunto(s)
Helianthus/microbiología , Oomicetos/patogenicidad , Fungicidas Industriales/farmacología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , VirulenciaRESUMEN
Major gene resistance to sunflower downy mildew (Plasmopara halstedii) races 304 and 314 was found to segregate independently from the resistance to races 334, 307 and 304 determined by the gene Pl2, already positioned on Linkage Group (LG) 8 of sunflower molecular maps. Using a consensus SSR-SNP map constructed from the INEDI RIL population and a new RIL population FU × PAZ2, the positions of Pl2 and Pl5 were confirmed and the new gene, denoted Pl21, was mapped on LG13, at 8 cM from Pl5. The two RIL populations were observed for their quantitative resistance to downy mildew in the field and both indicated the existence of a QTL on LG8 at 20-40 cM from the major resistance gene cluster. In addition, for the INEDI population, a strong QTL on LG10, reported previously, was confirmed and a third QTL was mapped on LG7. A growth chamber test methodology, significantly correlated with field results, also revealed the major QTL on LG10, explaining 65 % of variability. This QTL mapped in the same area as a gene involved in stomatal opening and root growth, which may be suggested as a possible candidate to explain the control of this character. These results indicate that it should be possible to combine major genes and other resistance mechanisms, a strategy that could help to improve durability of sunflower resistance to downy mildew.
Asunto(s)
Mapeo Cromosómico , Genes de Plantas/genética , Helianthus/genética , Helianthus/microbiología , Inmunidad Innata/genética , Oomicetos/patogenicidad , Enfermedades de las Plantas/genética , Sitios de Carácter Cuantitativo , Cromosomas de las Plantas/genética , ADN de Plantas/genética , Marcadores Genéticos , Genotipo , Helianthus/inmunología , Desequilibrio de Ligamiento , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genéticaRESUMEN
BACKGROUND: Downy mildew in sunflowers (Helianthus annuus L.) is caused by the oomycete Plasmopara halstedii (Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between H. annuus and P. halstedii have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of P. halstedii. RESULTS: A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of P. halstedii, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using in silico filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms. CONCLUSIONS: This study identified a large number of genes that are expressed during H. annuus/P. halstedii compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in P. halstedii similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of P. halstedii. This work therefore provides valuable tools for further discoveries regarding the H. annuus/P. halstedii pathosystem.
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Helianthus/genética , Oomicetos/genética , Polimorfismo de Nucleótido Simple , Transcriptoma , Secuencia de Aminoácidos , Análisis por Conglomerados , Mapeo Contig , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Helianthus/parasitología , Datos de Secuencia Molecular , Alineación de Secuencia , Factores de TiempoRESUMEN
This study aimed at defining the role of a basic helix-loop-helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria. We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics. We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor. Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Medicago truncatula/metabolismo , Rhizobium/fisiología , Nódulos de las Raíces de las Plantas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Medicago truncatula/genética , Medicago truncatula/microbiología , Medicago truncatula/fisiología , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Fenotipo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Rhizobium/genética , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Simbiosis/genéticaRESUMEN
Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.
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Proteínas Portadoras/fisiología , Medicago truncatula/microbiología , Medicago truncatula/fisiología , Fosfoproteínas/fisiología , Proteínas de Plantas/fisiología , Sinorhizobium meliloti/fisiología , Simbiosis/fisiología , Secuencia de Bases , Proteínas Portadoras/genética , Cartilla de ADN/genética , Medicago truncatula/genética , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Interferencia de ARN , Rhizobium/genética , Transducción de Señal , Transformación GenéticaRESUMEN
We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod- M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.
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Regulación de la Expresión Génica de las Plantas , Medicago truncatula/genética , Hibridación de Ácido Nucleico/métodos , Sinorhizobium meliloti/crecimiento & desarrollo , Simbiosis/genética , ADN Complementario/química , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Genes de Plantas , Medicago truncatula/microbiología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genéticaRESUMEN
In this study, we describe a large-scale expression-profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti. Macro- and microarrays containing about 6,000 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions. The experiments performed on wild-type and symbiotic mutant material led us to identify a set of 756 genes either up- or down-regulated at different stages of the nodulation process. Among these, 41 known nodulation marker genes were up-regulated as expected, suggesting that we have identified hundreds of new nodulation marker genes. We discuss the possible involvement of this wide range of genes in various aspects of the symbiotic interaction, such as bacterial infection, nodule formation and functioning, and defense responses. Importantly, we found at least 13 genes that are good candidates to play a role in the regulation of the symbiotic program. This represents substantial progress toward a better understanding of this complex developmental program.
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Genes de Plantas , Medicago truncatula/genética , Transporte Biológico , Pared Celular/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/metabolismo , Medicago truncatula/microbiología , Fijación del Nitrógeno , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Proteómica , Transducción de Señal , Sinorhizobium meliloti/fisiología , Simbiosis/fisiología , Transcripción GenéticaRESUMEN
Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.
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Proteínas de Arabidopsis/genética , Arabidopsis/microbiología , Cromosomas de las Plantas , Enfermedades de las Plantas/microbiología , Proteínas Quinasas/genética , Ralstonia solanacearum/patogenicidad , Arabidopsis/enzimología , Arabidopsis/genética , Mapeo Cromosómico , Genoma de Planta , Inmunidad Innata , Ralstonia solanacearum/clasificación , SerotipificaciónRESUMEN
We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface.