RESUMEN
Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development, and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results showed that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Infertilidad Vegetal , Polen , Polen/genética , Polen/crecimiento & desarrollo , Infertilidad Vegetal/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Genes MitocondrialesRESUMEN
BACKGROUND: The floral transition is a complex developmental event, fine-tuned by various environmental and endogenous cues to ensure the success of offspring production. Leaves are key organs in sensing floral inductive signals, such as a change in light regime, and in the production of the mobile florigen. CONSTANS and FLOWERING LOCUS T are major players in leaves in response to photoperiod. Morphological and molecular events during the floral transition have been intensively studied in the shoot apical meristem. To better understand the concomitant processes in leaves, which are less described, we investigated the nuclear changes in fully developed leaves during the time course of the floral transition. RESULTS: We highlighted new putative regulatory candidates of flowering in leaves. We observed differential expression profiles of genes related to cellular, hormonal and metabolic actions, but also of genes encoding long non-coding RNAs and new natural antisense transcripts. In addition, we detected a significant increase in ploidy level during the floral transition, indicating endoreduplication. CONCLUSIONS: Our data indicate that differentiated mature leaves, possess physiological plasticity and undergo extensive nuclear reprogramming during the floral transition. The dynamic events point at functionally related networks of transcription factors and novel regulatory motifs, but also complex hormonal and metabolic changes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reprogramación Celular/genética , Endorreduplicación/genética , Florigena/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Flores/fisiología , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Meristema/efectos de la radiación , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Structural variation is a major source of genetic diversity and an important substrate for selection. In allopolyploids, homoeologous exchanges (i.e. between the constituent subgenomes) are a very frequent type of structural variant. However, their direct impact on gene content and gene expression had not been determined. Here, we used a tissue-specific mRNA-Seq dataset to measure the consequences of homoeologous exchanges (HE) on gene expression in Brassica napus, a representative allotetraploid crop. We demonstrate that expression changes are proportional to the change in gene copy number triggered by the HEs. Thus, when homoeologous gene pairs have unbalanced transcriptional contributions before the HE, duplication of one copy does not accurately compensate for loss of the other and combined homoeologue expression also changes. These effects are, however, mitigated over time. This study sheds light on the origins, timing and functional consequences of homeologous exchanges in allopolyploids. It demonstrates that the interplay between new structural variation and the resulting impacts on gene expression, influences allopolyploid genome evolution.
Asunto(s)
Brassica napus/genética , Dosificación de Gen , Variación Genética , Genoma de Planta/genética , Expresión Génica , Especificidad de Órganos , Poliploidía , Recombinación Genética , Análisis de Secuencia de ARNRESUMEN
Cell wall remodeling is an essential mechanism for the regulation of plant growth and architecture, and xyloglucans (XyGs), the major hemicellulose, are often considered as spacers of cellulose microfibrils during growth. In the seed, the activity of cell wall enzymes plays a critical role in germination by enabling embryo cell expansion leading to radicle protrusion, as well as endosperm weakening prior to its rupture. A screen for Arabidopsis (Arabidopsis thaliana) mutants affected in the hormonal control of germination identified a mutant, xyl1, able to germinate on paclobutrazol, an inhibitor of gibberellin biosynthesis. This mutant also exhibited reduced dormancy and increased resistance to high temperature. The XYL1 locus encodes an α-xylosidase required for XyG maturation through the trimming of Xyl. The xyl1 mutant phenotypes were associated with modifications to endosperm cell wall composition that likely impact on its resistance, as further demonstrated by the restoration of normal germination characteristics by endosperm-specific XYL1 expression. The absence of phenotypes in mutants defective for other glycosidases, which trim Gal or Fuc, suggests that XYL1 plays the major role in this process. Finally, the decreased XyG abundance in hypocotyl longitudinal cell walls of germinating embryos indicates a potential role in cell wall loosening and anisotropic growth together with pectin de-methylesterification.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Glucanos/metabolismo , Xilanos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Endospermo/crecimiento & desarrollo , Endospermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Germinación/efectos de los fármacos , Germinación/genética , Germinación/fisiología , Mutación , Plantas Modificadas Genéticamente , Procesamiento Proteico-Postraduccional , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Triazoles/farmacología , Xilosidasas/genética , Xilosidasas/metabolismoRESUMEN
Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.
Asunto(s)
Magnoliopsida/genética , Meiosis , Evolución Molecular , Duplicación de Gen , Genoma de Planta , Recombinación HomólogaRESUMEN
The Unfolded Protein Response (UPR) is a retrograde, ER-to-nucleus, signalling pathway which is conserved across kingdoms. In plants, it contributes to development, reproduction, immunity and tolerance to abiotic stress. This RNA sequencing (RNA-seq) dataset was produced from 14-day-old Arabidopsis thaliana seedlings challenged by tunicamycin (Tm), an antibiotic inhibiting Asn-linked glycosylation in the endoplasmic reticulum (ER), causing an ER stress and eventually activating the UPR. Wild-type (WT) and a double mutant deficient for two main actors of the UPR (INOSITOL-REQUIRING ENZYME 1A and INOSITOL-REQUIRING ENZYME 1B) were used as genetic backgrounds in our experimental setup, allowing to distinguish among differentially-expressed genes (DEGs) which ones are dependent on or independent on IRE1s. Also, shoots and roots were harvested separately to determine organ-specific transcriptomic responses to Tm. Library and sequencing were performed using DNBseq™ technology by the Beijing Genomics Institute. Reads were mapped and quantified against the Arabidopsis genome. Differentially-expressed genes were identified using Rflomics upon filtering and normalization by the Trimmed Mean of M-value (TMM) method. While the genotype effect was weak under mock conditions (with a total of 182 DEGs in shoots and 195 DEGs in roots), the tunicamycin effect on each genotype was characterized by several hundred of DEGs in both shoots and roots. Among these genes, 872 and 563 genes were statistically up- and down-regulated in the shoot tissues of the double mutant when compared to those of WT, respectively. In roots of Tm-challenged seedlings, 425 and 439 genes were significantly up- and down-regulated in mutants with respect to WT. We believe that our dataset could be reused for investigating any biological questions linked to ER homeostasis and its role in plant physiology.
RESUMEN
Meiotic rapid prophase chromosome movements (RPMs) require connections between the chromosomes and the cytoskeleton, involving SUN (Sad1/UNC-84)-domain-containing proteins at the inner nuclear envelope (NE). RPMs remain significantly understudied in plants, with respect to their importance in the regulation of meiosis. Here, we demonstrate that Arabidopsis thaliana meiotic centromeres undergo rapid (up to 500 nm/s) and uncoordinated movements during the zygotene and pachytene stages. These centromere movements are not affected by altered chromosome organization and recombination but are abolished in the double mutant sun1 sun2. We also document the changes in chromosome dynamics and nucleus organization during the transition from leptotene to zygotene, including telomere attachment to SUN-enriched NE domains, bouquet formation, and nucleolus displacement, all of which were defective in sun1 sun2. These results establish A. thaliana as a model species for studying the functional implications of meiotic RPMs and demonstrate the mechanistic conservation of telomere-led RPMs in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Cromosomas de las Plantas , Meiosis , Membrana Nuclear , Telómero , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromosomas de las Plantas/genética , Telómero/metabolismo , Centrómero/metabolismo , Profase , Profase Meiótica I , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMEN
BACKGROUND: Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently. RESULTS: To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of wpgrp2, wpgrp3, coleoptericin-B, diptericin, and sarcotoxin genes in aposymbiotic insects. CONCLUSIONS: Library sequencing significantly increased the number of unigenes, allowing for improved functional and genetic investigations in the cereal weevil S. oryzae. Transcriptomic analyses support selective and local immune gene expression in the bacteriome tissue and uncover cellular pathways that are of potential interest to bacteriocyte survival and homeostasis. Bacterial challenge experiments have revealed that the systemic immune response would be less induced in a symbiotic insect, thus highlighting new perspectives on host immunity in long-term invertebrate co-evolutionary associations.
Asunto(s)
Gammaproteobacteria/fisiología , Genómica/métodos , Proteínas de Insectos/genética , Gorgojos/genética , Animales , Proteínas Bacterianas/genética , Regulación de la Expresión Génica , Biblioteca de Genes , Especificidad del Huésped , Interacciones Huésped-Patógeno , Larva/microbiología , Microbiota , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Simbiosis , Gorgojos/embriología , Gorgojos/microbiología , Gorgojos/fisiologíaRESUMEN
BACKGROUND: Wolbachia are intracellular bacteria known to be facultative reproductive parasites of numerous arthropod hosts. Apart from these reproductive manipulations, recent findings indicate that Wolbachia may also modify the host's physiology, notably its immune function. In the parasitoid wasp, Asobara tabida, Wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. The absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of Wolbachia in the wasp could also be responsible for this dependence. In order to decipher the general mechanisms underlying host-Wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals. RESULTS: As no genetic data were available on A. tabida, we constructed several Expressed Sequence Tags (EST) libraries, and obtained 12,551 unigenes from this species. Gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction (SSH). As pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death (PCD) and immunity (broad sense) was analyzed by quantitative RT-PCR. We showed that Wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation. We also showed that Wolbachia may interact with immune gene expression to ensure its persistence within the host. CONCLUSIONS: This study allowed us to constitute the first major dataset of the transcriptome of A. tabida, a species that is a model system for both host/Wolbachia and host/parasitoid interactions. More specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of Wolbachia should also be investigated beyond reproductive manipulations.
Asunto(s)
Regulación de la Expresión Génica , Genes de Insecto , Ovario/metabolismo , Avispas/genética , Wolbachia/fisiología , Animales , Femenino , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Masculino , Ovario/microbiología , Simbiosis , Avispas/inmunología , Avispas/microbiología , Avispas/fisiologíaRESUMEN
BACKGROUND: Wolbachia are vertically transmitted bacteria known to be the most widespread endosymbiont in arthropods. They induce various alterations of the reproduction of their host, including feminization of genetic males in isopod crustaceans. In the pill bug Armadillidium vulgare, the presence of Wolbachia is also associated with detrimental effects on host fertility and lifespan. Deleterious effects have been demonstrated on hemocyte density, phenoloxidase activity, and natural hemolymph septicemia, suggesting that infected individuals could have defective immune capacities. Since nothing is known about the molecular mechanisms involved in Wolbachia-A. vulgare interactions and its secondary immunocompetence modulation, we developed a transcriptomics strategy and compared A. vulgare gene expression between Wolbachia-infected animals (i.e., "symbiotic" animals) and uninfected ones (i.e., "asymbiotic" animals) as well as between animals challenged or not challenged by a pathogenic bacteria. RESULTS: Since very little genetic data is available on A. vulgare, we produced several EST libraries and generated a total of 28 606 ESTs. Analyses of these ESTs revealed that immune processes were over-represented in most experimental conditions (responses to a symbiont and to a pathogen). Considering canonical crustacean immune pathways, these genes encode antimicrobial peptides or are involved in pathogen recognition, detoxification, and autophagy. By RT-qPCR, we demonstrated a general trend towards gene under-expression in symbiotic whole animals and ovaries whereas the same gene set tends to be over-expressed in symbiotic immune tissues. CONCLUSION: This study allowed us to generate the first reference transcriptome ever obtained in the Isopoda group and to identify genes involved in the major known crustacean immune pathways encompassing cellular and humoral responses. Expression of immune-related genes revealed a modulation of host immunity when females are infected by Wolbachia, including in ovaries, the crucial tissue for the Wolbachia route of transmission.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Isópodos/genética , Isópodos/inmunología , Wolbachia/fisiología , Animales , Etiquetas de Secuencia Expresada , Femenino , Regulación de la Expresión Génica , Isópodos/microbiología , Isópodos/fisiología , Masculino , Ovario/inmunología , Ovario/metabolismo , Simbiosis , Wolbachia/inmunologíaRESUMEN
CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.
Asunto(s)
Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma de Planta/genética , Plantas/genética , Plantas Modificadas Genéticamente/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
BACKGROUND: The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. RESULTS: A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO) terms, and thousands of single-nucleotide polymorphisms (SNPs) were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49%) that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. CONCLUSION: The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to further develop Silene as a plant model system. The genes characterized will be useful for future research not only in the species included in the present study, but also in related species for which no genomic resources are yet available. Our results demonstrate the efficiency of massively parallel transcriptome sequencing in a comparative framework as an approach for developing genomic resources in diverse groups of non-model organisms.
Asunto(s)
Bases de Datos Genéticas , Etiquetas de Secuencia Expresada/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular/métodos , Silene/genética , Transcriptoma/genética , Dianthus/genética , Fenómenos Ecológicos y Ambientales , Evolución Molecular , Tamaño del Genoma , Heterocigoto , Polimorfismo de Nucleótido Simple/genética , Reproducibilidad de los Resultados , Homología de Secuencia de Ácido NucleicoRESUMEN
Wolbachia is an intracellular bacterium generally described as being a facultative reproductive parasite. However, Wolbachia is necessary for oogenesis completion in the wasp Asobara tabida. This dependence has evolved recently as a result of interference with apoptosis during oogenesis. Through comparative transcriptomics between symbiotic and aposymbiotic individuals, we observed a differential expression of ferritin, which forms a complex involved in iron storage. Iron is an essential element that is in limited supply in the cell. However, it is also a highly toxic precursor of Reactive Oxygen Species (ROS). Ferritin has also been shown to play a key role in host-pathogen interactions. Measuring ferritin by quantitative RT-PCR, we confirmed that ferritin was upregulated in aposymbiotic compared to symbiotic individuals. Manipulating the iron content in the diet, we showed that iron overload markedly affected wasp development and induced apoptotic processes during oogenesis in A. tabida, suggesting that the regulation of iron homeostasis may also be related to the obligate dependence of the wasp. Finally, we demonstrated that iron metabolism is influenced by the presence of Wolbachia not only in the obligate mutualism with A. tabida, but also in facultative parasitism involving Drosophila simulans and in Aedes aegypti cells. In these latter cases, the expression of Wolbachia bacterioferritin was also increased in the presence of iron, showing that Wolbachia responds to the concentration of iron. Our results indicate that Wolbachia may generally interfere with iron metabolism. The high affinity of Wolbachia for iron might be due to physiological requirement of the bacterium, but it could also be what allows the symbiont to persist in the organism by reducing the labile iron concentration, thus protecting the cell from oxidative stress and apoptosis. These findings also reinforce the idea that pathogenic, parasitic and mutualistic intracellular bacteria all use the same molecular mechanisms to survive and replicate within host cells. By impacting the general physiology of the host, the presence of a symbiont may select for host compensatory mechanisms, which extends the possible consequences of persistent endosymbiont on the evolution of their hosts.
Asunto(s)
Ferritinas/genética , Insectos/genética , Insectos/metabolismo , Hierro/metabolismo , Wolbachia/fisiología , Aedes/genética , Aedes/crecimiento & desarrollo , Aedes/metabolismo , Aedes/microbiología , Animales , Secuencia de Bases , Mapeo Cromosómico , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Drosophila/microbiología , Femenino , Ferritinas/metabolismo , Regulación de la Expresión Génica , Sitios Genéticos , Interacciones Huésped-Parásitos/genética , Insectos/crecimiento & desarrollo , Insectos/microbiología , Hierro/farmacología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Infecciones por Rickettsiaceae/genética , Infecciones por Rickettsiaceae/metabolismo , Simbiosis/genética , Avispas/genética , Avispas/crecimiento & desarrollo , Avispas/metabolismo , Avispas/microbiología , Wolbachia/genéticaRESUMEN
The seeds of Arabidopsis thaliana become encapsulated by a layer of mucilage when imbibed. This polysaccharide-rich hydrogel is constituted of two layers, an outer layer that can be easily extracted with water and an inner layer that must be examined in situ in order to study its properties and structure in a non-destructive manner or disintegrated through hydrolysis or physical means in order to analyze its constituents. Mucilage production is an adaptive trait and we have exploited 19 natural accessions previously found to have atypical and varied outer mucilage characteristics. A detailed study using biochemical, histological and Time-Domain NMR analyses has been used to generate three related datasets covering 33 traits measured in four biological replicates. This data will be a rich resource for genetic, biochemical, structural and functional analyses investigating mucilage constituent polysaccharides or their role as adaptive traits.
Asunto(s)
Arabidopsis/genética , Polisacáridos/genética , Semillas/química , Regulación de la Expresión Génica de las Plantas , Semillas/genéticaRESUMEN
Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA-based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont-host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.
Asunto(s)
Bacterias/clasificación , Evolución Biológica , Hemípteros/genética , Hemípteros/microbiología , Filogenia , Simbiosis , Animales , Bacterias/genética , ADN Mitocondrial/genética , ADN Espaciador Ribosómico/genética , Desequilibrio de Ligamiento , Datos de Secuencia MolecularRESUMEN
This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.
Asunto(s)
Brassica/genética , Miosis , Mutación , Poliploidía , Sistemas CRISPR-Cas , Edición Génica , Genoma de Planta , Genotipo , Recombinación Genética , Análisis de Secuencia de ADN , Transformación GenéticaRESUMEN
[This corrects the article DOI: 10.3389/fpls.2018.00368.].
RESUMEN
For insects, the prevalence of numerous vertically transmitted viruses can be high in their host populations. These viruses often have few, if any, pathological effects on their hosts, and consequently, many of them can remain unnoticed for long periods, despite their potential role in the evolution of the host phenotype. Some females of Leptopilina boulardi, a solitary parasitoid of Drosophila larvae, are infected by an inherited virus (LbFV) that manipulates the behavior of the wasp by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). This behavioral alteration allows horizontal transmission of the virus within superparasitized Drosophila larvae. Using suppressive subtractive hybridization with infected and uninfected lines, we identified one putative viral sequence. Based on this sequence, we developed a simple PCR test. We tested the correlation between the superparasitism phenotype and PCR amplification of the putative viral marker using several experimental conditions (including horizontal transfers) and several parasitoid genotypes. All of the results revealed that there was a perfect match between the superparasitism phenotype and the amplification profile, which validated use of the molecular marker as a tool to track the presence of the virus and provided the first genomic data for this fascinating virus. The results also show that there was very efficient horizontal and vertical transmission of LbFV, which probably explains its high prevalence in the French populations that we sampled (67 and 70% of infected females). This manipulative virus is likely to play a major role in the ecology and evolution of its parasitoid host.
Asunto(s)
Conducta Animal , Himenópteros/virología , Virosis/fisiopatología , Virus/aislamiento & purificación , Virus/patogenicidad , Animales , Transmisión de Enfermedad Infecciosa , Virus/genéticaRESUMEN
BACKGROUND: Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome). RESULTS: To study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome. CONCLUSION: The current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Insectos/genética , Alphaproteobacteria/fisiología , Animales , Péptidos Catiónicos Antimicrobianos/genética , Proteínas Portadoras/genética , Escherichia coli/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Larva/inmunología , Larva/microbiología , Datos de Secuencia Molecular , Muramidasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Gorgojos/genética , Gorgojos/inmunología , Gorgojos/microbiologíaRESUMEN
UNLABELLED: Comparing genetic and physical maps (the so-called Marey map approach) is still the most widely used approach to estimate genome-wide recombination rates. Remarkably, there is no available bioinformatics tool specifically devoted to Marey map approach. Here, we developed such a tool called MareyMap based on GNU R and Tcl/Tk. MareyMap offers a user-friendly graphical interface and includes useful features, such as data cleaning process, sophisticated interpolation methods to estimate local rates, possibility of complex queries, various range of import-export files. Moreover, MareyMap comes with ready-to-use maps for human, Drosophila, Caenorhabditis elegans and Arabidopsis. MareyMap has been made so that it can be easily upgraded with new data and interpolation methods. AVAILABILITY: http://pbil.univ-lyon1.fr/software/mareymap/.