A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94 Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5 kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant–pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes

Detection of non-coding RNA in bacteria and archaea using the DETR'PROK Galaxy pipeline

International audienceRNA-seq experiments are now routinely used for the large scale sequencing of transcripts. In bacteria or archaea, such deep sequencing experiments typically produce 10-50 million fragments that cover most of the genome, including intergenic regions. In this context, the precise delineation of the non-coding elements is challenging. Non-coding elements include untranslated regions (UTRs) of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the antisense strand of genes (asRNA). Here we present a computational pipeline (DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework that takes as input a mapping of deep sequencing reads and performs successive steps of clustering, comparison with existing annotation and identification of transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and asRNAs. We provide a step-by-step description of the protocol using real-life example data sets from Vibrio splendidus and Escherichia coil. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved

Genome-wide Translational Changes Induced by the Prion [PSI+]

SummaryPrions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI+] is an aggregated conformation of the translational release factor eRF3. [PSI+] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI+]-controlled expression remain unknown. We used ribosome profiling in isogenic [PSI+] and [psi−] strains to identify the changes induced by [PSI+]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI+]. Surprisingly, [PSI+] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI+] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi−] and [PSI+] strains

Analyse transcriptomique cellule-unique pour définir les populations cellulaires pluripotentes et extra-embryonnaires et leurs interactions dans le blastocyste porcin

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Volume reduction and automation of DNAprep libraries for low pass sequencing

International audienceTo characterize a population's haplotypic variability, low pass sequencing represents an interesting alternative to genotyping DNA-microarrays. Although this new technology is also costly, it offers greater precision of genetic information. We have therefore reduced reagent and DNA volumes by automating our Illumina DNA Prep library preparation protocol with the Mosquito HV (SPT LabTech, UK). For this purpose, we intend to evaluate this strategy for the porcine species. We used a family F2 protocol based on crossbreeds between Large White (LW) and Meishan (MS) individuals. It comprises around 1000 pigs, including 12 F0 (6 LW and 6 MS), 30 F1 (6 males and 24 females) and 1000 F2. In addition to the evaluation of bioinformatic phasing methods for the imputation and reconstruction of haplotypes. The exploitation of these data will enable the precise characterization of recombination sites. This will provide an understanding of the reasons why a large region of the X chromosome recombines or not. The SeqOccIn porcine pilot project gave us the opportunity to (i) automate the DNA prep protocol to significantly reduce volumes with very good results and (ii) generate good quality low pass sequencing data for genotype imputation (validated by Mendelian compatibility

Genome sequence and annotation of Periconia digitata a hopeful biocontrol agent of phytopathogenic oomycetes

Abstract The Periconia fungal genus belongs to the phylum Ascomycota, order Pleosporales, family Periconiaceae. Periconia are found in many habitats, but little is known about their ecology. Several species from this genus produce bioactive molecules. Periconia digitata extracts were shown to be deadly active against the pine wilt nematode. Furthermore, P. digitata was shown to inhibit the plant pathogenic oomycete Phytophthora parasitica. Because P. digitata has great potential as a biocontrol agent and high quality genomic resources are still lacking in the Periconiaceae family, we generated long-read genomic data for P. digitata. Using PacBio Hifi sequencing technology, we obtained a highly-contiguous genome assembled in 13 chromosomes and totaling ca. 39 Mb. In addition, we produced a reference transcriptome, based on 12 different culture conditions, and proteomic data to support the genome annotation. Besides representing a new reference genome within the Periconiaceae, this work will contribute to our better understanding of the Eukaryotic tree of life and opens new possibilities in terms of biotechnological applications

Maintaining Two Mating Types: Structure of the Mating Type Locus and Its Role in Heterokaryosis in Podospora anserina

International audiencePseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat- isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat- mating types of P. anserina, which is mostly found in nature as a mat+/mat- heterokaryotic mycelium harboring sexually compatible nuclei. We identified a "mat" region ∼0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat- strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat- chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat-, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat-/mat- heterokaryons are as stable as mat+/mat- ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat- strains

Dynamic stability of the cyathostomin – gut microbiota interactions in horses

International audienceThe dynamics of the helminth-microbiota assemblage in their host remain largely unexplored. Here, we used the horse – cyathostomin system to quantify the resilience of the nematode-bacteria interactions following an anthelmintic treatment. Ten infected Welsh ponies received a pyrantel treatment to eliminate adult parasites in the gut lumen. These were matched with uninfected treated individuals to isolate the treatment effect. These two groups were matched with untreated control individuals. Metabarcoding approaches were implemented to track faecal microbiota and cyathostomin community compositions over a 42-day time-course.The nemabiome approach identified 13 species overwhelmed by the abundance of Cylicocyclus nassatus that accounted for 52.6% of the overall diversity. The gut microbiota of infected horses exhibited higher Shannon entropy and bacterial species turnover, suggesting orderly rearrangements of assemblages. Enterococcus abundance could however discriminate between infected and uninfected ponies.Following pyrantel treatment, the dynamic stability of bacterial community in treated horses chiefly increased towards unstability but reached a stasis with limited variation across ponies afterwards. This would be compatible with creation of a new equilibrium between bacterial genera upon pyrantel treatment. Using a convergent cross-mapping approach, we evidenced a set of core bacterial genera, i.e. Fibrobacter, Saccharofermentans, and Aloprevotella that likely provide the stabilizing forces towards this new equilibrium. The cyathostomin community recovered quickly with a primarily unchanged structure 42 days after treatment.Our data provide the first description of the resilience of the horse gut microbiota in infected horses, indicating a heavily canalized system

The characterization of novel tissue microbiota using an optimized 16S metagenomic sequencing pipeline

Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S meta-barcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease