RESUMEN
We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.
Asunto(s)
Resistencia a la Enfermedad , Evolución Molecular , Glycine max/genética , Familia de Multigenes , Phaseolus/genética , Secuencia de Aminoácidos , Teorema de Bayes , Diploidia , Genes de Plantas , Phaseolus/química , Phaseolus/inmunología , Phaseolus/microbiología , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Dominios y Motivos de Interacción de Proteínas , Pseudomonas/inmunología , Pseudomonas/patogenicidad , Recombinación Genética , Selección Genética , Alineación de Secuencia , Glycine max/química , Glycine max/inmunología , Glycine max/microbiología , TetraploidíaRESUMEN
A pathosystem between Aphanomyces euteiches, the causal agent of pea root rot disease, and the model legume Medicago truncatula was developed to gain insights into mechanisms involved in resistance to this oomycete. The F83005.5 French accession and the A17-Jemalong reference line, susceptible and partially resistant, respectively, to A. euteiches, were selected for further cytological and genetic analyses. Microscopy analyses of thin root sections revealed that a major difference between the two inoculated lines occurred in the root stele, which remained pathogen free in A17. Striking features were observed in A17 roots only, including i) frequent pericycle cell divisions, ii) lignin deposition around the pericycle, and iii) accumulation of soluble phenolic compounds. Genetic analysis of resistance was performed on an F7 population of 139 recombinant inbred lines and identified a major quantitative trait locus (QTL) near the top of chromosome 3. A second study, with near-isogenic line responses to A. euteiches confirmed the role of this QTL in expression of resistance. Fine-mapping allowed the identification of a 135-kb sequenced genomic DNA region rich in proteasome-related genes. Most of these genes were shown to be induced only in inoculated A17. Novel mechanisms possibly involved in the observed partial resistance are proposed.
Asunto(s)
Aphanomyces/fisiología , Medicago truncatula/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Raíces de Plantas/microbiología , Complejo de la Endopetidasa Proteasomal/genética , Sitios de Carácter Cuantitativo/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Inmunidad Innata/genética , Endogamia , Medicago truncatula/citología , Medicago truncatula/genética , Mapeo Físico de Cromosoma , Enfermedades de las Plantas/microbiología , Raíces de Plantas/citología , Raíces de Plantas/genética , Reproducibilidad de los ResultadosRESUMEN
Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.
Asunto(s)
Evolución Molecular , Glycine max/genética , Retroelementos , Secuencia de Bases , ADN de Plantas/química , Eliminación de Gen , Genoma de Planta , Genómica/métodos , Elementos de Nucleótido Esparcido Largo , Metilación , Mutagénesis Insercional , Phaseolus/genética , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Secuencias Repetidas TerminalesRESUMEN
The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.
Asunto(s)
Evolución Molecular , Duplicación de Gen , Genes de Plantas , Glycine max/genética , Poliploidía , Retroelementos , Centrómero/genética , Cromosomas Artificiales Bacterianos , ADN de Plantas/química , Eliminación de Gen , Genoma de Planta , Inmunidad Innata/genética , Familia de Multigenes , Mutagénesis Insercional , Phaseolus/genética , Filogenia , Enfermedades de las Plantas/genética , Análisis de Secuencia de ADNRESUMEN
Medicago truncatula was used to characterize resistance to anthracnose and powdery mildew caused by Colletotrichum trifolii and Erysiphe pisi, respectively. Two isolates of E. pisi (Ep-p from pea and Ep-a from alfalfa) and two races of C. trifolii (races 1 and 2) were used in this study. The A17 genotype was resistant and displayed a hypersensitive response after inoculation with either pathogen, while lines F83005.5 and DZA315.16 were susceptible to anthracnose and powdery mildew, respectively. To identify the genetic determinants underlying resistance in A17, two F7 recombinant inbred line (RIL) populations, LR4 (A17 x DZA315.16) and LR5 (A17 x F83005.5), were phenotyped with E. pisi isolates and C. trifolii races, respectively. Genetic analyses showed that i) resistance to anthracnose is governed mainly by a single major locus to both races, named Ct1 and located on the upper part of chromosome 4; and ii) resistance to powdery mildew involves three distinct loci, Epp1 on chromosome 4 and Epa1 and Epa2 on chromosome 5. The use of a consensus genetic map for the two RIL populations revealed that Ct1 and Epp1, although located in the same genome region, were clearly distinct. In silico analysis in this region identified the presence of several clusters of nucleotide binding site leucine-rich repeat genes. Many of these genes have atypical resistance gene analog structures and display differential expression patterns in distinct stress-related cDNA libraries.
Asunto(s)
Ascomicetos/fisiología , Colletotrichum/fisiología , Inmunidad Innata/genética , Medicago truncatula/genética , Medicago truncatula/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Cromosomas de las Plantas , Etiquetas de Secuencia Expresada , Genes de Plantas , Marcadores Genéticos , Genotipo , Inmunidad Innata/inmunología , Endogamia , Medicago truncatula/inmunología , Medicago truncatula/ultraestructura , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Hojas de la Planta/ultraestructura , Sitios de Carácter CuantitativoRESUMEN
The nucleotide-binding site (NBS)-Leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes, and is one of the largest gene families in plant genomes. We have identified 333 nonredundant NBS-LRRs in the current Medicago truncatula draft genome (Mt1.0), likely representing 400 to 500 NBS-LRRs in the full genome, or roughly 3 times the number present in Arabidopsis (Arabidopsis thaliana). Although many characteristics of the gene family are similar to those described on other plant genomes, several evolutionary features are particularly pronounced in M. truncatula, including a high degree of clustering, evidence of significant numbers of ectopic translocations from clusters to other parts of the genome, a small number of more evolutionarily stable NBS-LRRs, and numerous truncations and fusions leading to novel domain compositions. The gene family clearly has had a large impact on the structure of the genome, both through ectopic translocations (potentially, a means of seeding new NBS-LRR clusters), and through two extraordinarily large superclusters. Chromosome 6 encodes approximately 34% of all TIR-NBS-LRRs, while chromosome 3 encodes approximately 40% of all coiled-coil-NBS-LRRs. Almost all atypical domain combinations are in the TIR-NBS-LRR subfamily, with many occurring within one genomic cluster. This analysis shows the gene family not only is important functionally and agronomically, but also plays a structural role in the genome.
Asunto(s)
Genes de Plantas/genética , Medicago truncatula/genética , Secuencias de Aminoácidos , Análisis por Conglomerados , Computadores , Elementos Transponibles de ADN , Etiquetas de Secuencia Expresada , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Genoma de Planta , Filogenia , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Seudogenes/genéticaRESUMEN
The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.
Asunto(s)
Proteínas Algáceas/química , Pared Celular/química , Celulosa/metabolismo , Lectinas/química , Phytophthora/química , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Lectinas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Phytophthora/patogenicidad , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Nicotiana/anatomía & histología , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.