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Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteases specifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skin infections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis. In the present study, we characterized the new et gene at a genetic level and the enzymatic activity of the deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with other publicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%, 53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene shared the same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinant enzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene was thus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded the extracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canal epithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human and swine Dsg1 as well as murine Dsg1α and Dsg1β, but not canine Dsg1 or murine Dsg1γ. Molecular modeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavage site and species-specific cleavage activity, suggesting that the docking step preceding cleavage accounts for the ETE species-specificity. This new virulence factor may contribute to the bacterial colonization on the stratified epithelia in certain ruminants with mastitis.

Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre‐industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation., see also the Perspective by Brigida Gallone, Jan Steensels and Kevin J. Verstrepen.

Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteasesspecifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skininfections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis.In the present study, we characterized the new et gene at a genetic level and the enzymatic activity ofthe deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with otherpublicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%,53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene sharedthe same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinantenzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene wasthus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded theextracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canalepithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human andswine Dsg1 as well as murine Dsg1α and Dsg1β, but not canine Dsg1 or murine Dsg1γ. Molecularmodeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavagesite and species-specific cleavage activity, suggesting that the docking step preceding cleavageaccounts for the ETE species-specificity. This new virulence factor may contribute to the bacterialcolonization on the stratified epithelia in certain ruminants with mastitis.

International audience; Fungi are evolutionary shape shifters and adapt quickly to new environments. Ectomycorrhizal (EM) symbioses are mutualistic associations between fungi and plants and have evolved repeatedly and independently across the fungal tree of life, suggesting lineages frequently reconfigure genome content to take advantage of open ecological niches. To date analyses of genomic mechanisms facilitating EM symbioses have involved comparisons of distantly related species, but here, we use the genomes of three EM and two asymbiotic (AS) fungi from the genus Amanita as well as an AS outgroup to study genome evolution following a single origin of symbiosis. Our aim was to identify the defining features of EM genomes, but our analyses suggest no clear differentiation of genome size, gene repertoire size, or transposable element content between EM and AS species. Phylogenetic inference of gene gains and losses suggests the transition to symbiosis was dominated by the loss of plant cell wall decomposition genes, a confirmation of previous findings. However, the same dynamic defines the AS species A. inopinata, suggesting loss is not strictly associated with origin of symbiosis. Gene expansions in the common ancestor of EM Amanita were modest, but lineage specific and large gene family expansions are found in two of the three EM extant species. Even closely related EM genomes appear to share few common features. The genetic toolkit required for symbiosis appears already encoded in the genomes of saprotrophic species, and this dynamic may explain the pervasive, recurrent evolution of ectomycorrhizal associations.

[YG: Invited Keynote Lecture]; International audience

The study of the interactions between genetics and nutrition has gained new impetus with the development of high-throughput genomic approaches allowing for a comprehensive approach of the variability of gene sequences or activity. The object of nutrigenetics is to identify the genetic determinants (sequence polymorphisms) of the variability of individual responses to nutrition. A quantitative trait locus (QTL) is a chromosomal region involved in the control of quantitative phenotypic traits. Based on the knowledge of the chicken genome sequence, numerous positional candidate genes can be proposed within a QTL region, and further evaluation is needed to test the link between polymorphism of those genes, their activity and the expression of the phenotype. As a proof of principle that such an approach is relevant to nutrigenetics, we identified a polymorphism in the gene encoding the enzyme -carotene 15,15'-monooxygenase 1 (BCMO1), which affected the response to dietary intake of -carotene in term of plasma, liver and duodenal concentrations of lutein. Another project aims to identify the genetic determinants of digestive ability through the development and characterization of two experimental lines of chickens divergently selected for this trait. Recently, chromosomal regions controlling this trait have been identified, and further studies of sequence polymorphisms and gene expression (functional genomics) data in extreme animals issued from a cross population are underway. Eventually, the tools of nutrigenetics will predict the potential for adaptation of genotypes to various feeds. Nutrigenomics, allowing a better understanding of the mechanisms by which nutrition affects gene activity, is expected to propose new nutritional strategies to guide phenotypes and new selection strategies to improve birds’ ability to adapt to different diets. The combination of both approaches should permit to identify genes controlling those phenotypes.

Lignocellulosic biomass from cultivated poplars is a renewable resource of interest for the production of bio-energy. To improve this complex trait, it is essential to better understand the genetic factors that affect its variation. Within this context, to explore the components of genetic variability within a parental species of commercial hybrid poplars, we have initiated an integrative approach combining genomic, transcriptomic and phenotypic information in a large collection of individuals sampled from natural populations and raised in a clonal field trial.Here, we report the analysis of polymorphism within transcriptomic sequences produced by RNA-seq. RNA was extracted from young differentiating xylem and cambium collected on 24 3-years old trees corresponding to 2 replicates of 12 genotypes from 6 natural populations representative of the geographical distribution of the species in Western Europe. After RNA quantification, the RNA from xylem and cambium from the same tree were pooled and subjected to high-throughput sequencing on an Illumina HiSeq 2000 using 100 bp paired-end indexing runs. All the samples were sequenced on one Illumina flow-cell, yielding around 18 million of pairedend reads per sample.The sequences were first trimmed (quality and length) and aligned to the Populus trichocarpa v3 assembly using BWA-MEM. Around 99.7% of the reads mapped against the reference genome and 93.3% properly mapped despite the fact that the genome is from a different species. Then, we performed a mapping filtering for duplicated sequence reads after alignment to the genome with Picard tools to mitigate biases introduced by data generation steps such as PCR amplification. We also performed local realignment around indels and recalibrated the base quality scores with GATK. The variant discovery process was based on several variant callers (GATK, VarScan, FreeBayes) with several filtering parameters (VCF tools). We compared the genotype calls to those previously obtained with a SNP array (Faivre-Rampant et al, 2016) in order to choose the optimal variant caller and the SNP filtering parameters (depth, quality).The usefulness and relevance of the resulting SNPs for population genomic studies was further assessed by analyzing: (i) genomewide patterns of nucleotide diversity, (ii) population structure, and (iii) linkage disequilibrium. The SNPs have also been annotated in order to estimate Ka/Ks ratio, with a particular focus on candidate genes involved in the control of lignocellulosic biomass production and quality.This promising approach is currently being extended to 240 genotypes in order to validate the genomics patterns identified in the present study.

International audience; The diversity among rust fungi is simply astounding: over 7000 species that evolved to colonize niches all over the plant kingdom. This diversification likely involved major host jumps, especially considering that the life cycles of heteroecious rusts, such as the cereal rusts, involve sexual and asexual stages that take place on completely unrelated host plants (Savile, 1976; McTaggart et al., 2015), but also co-evolution in diverse natural settings, establishing equilibrium (e.g., Thrall et al., 2012). These interactions resulted in complex life styles including for many rusts, the production of up to five different spore types, each requiring very different developmental programs and hence gene expression.

International audience; Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the discovery of complex patterns in gene expression of soil fungal communities.

Next-generation sequencing (NGS) technologies have made high-throughput sequencing available to medium- and small-size laboratories, culminating in a tidal wave of genomic information. The quantity of sequenced bacterial genomes has not only brought excitement to the field of genomics but also heightened expectations that NGS would boost antibacterial discovery and vaccine development. Although many possible drug and vaccine targets have been discovered, the success rate of genome-based analysis has remained below expectations. Furthermore, NGS has had consequences for genome quality, resulting in an exponential increase in draft (partial data) genome deposits in public databases. If no further interests are expressed for a particular bacterial genome, it is more likely that the sequencing of its genome will be limited to a draft stage, and the painstaking tasks of completing the sequencing of its genome and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the "scientific value" of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses the factors that could be leading to the increase in the number of draft deposits and the consequent loss of relevant biological information.

L’évaluation pangénomique (GWE) est une méthodologie disruptive avec un potentiel d’augmentation des précisions de prédiction sans augmenter l’intervalle de génération. La recherche sur la GWE a été principalement focalisée sur les facteurs affectant la précision de prédiction. En comparaison, peu a été fait sur les impacts que cette précision supplémentaire pourrait avoir sur la diversité génétique à l'échelle du génome entier ou sur la façon d'utiliser efficacement la variation de la ségrégation Mendélienne (MS). Il existe des solutions, originalement développées pour la sélection sur pedigree, OCS et MA, mais elles reposent souvent sur des informations de consanguinité ou d’apparentement moyennes. Ainsi, l'information de la variation de la diversité au sein des génomes est négligée lors de la construction de telles solutions. L'objectif général est ici de concevoir un outil de sélection pour améliorer de manière cohérente le gain génétique et la gestion de la diversité avec GWE, en prenant en compte explicitement les informations de variation de diversité et MS le long du génome. Pour cela, nous utiliserons des approches de génétique quantitative, des simulations et des applications chez le peuplier, le maïs et le palmier à huile, pour lesquels le croisement est un élément clé de leur amélioration. Trois développements seront intégrés : (a) l'imputation qui devrait agir en synergie avec les deux autres ; (b) les modèles de prédiction avec des composantes non - additives pour prédire les croisements ; et (c) des outils pour la sélection et les croisements optimaux tout en respectant diversité et MS le long des génomes des individus sélectionnés.

Session 25. Free communications - molecular genetics; absent

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means., Author Summary Plant-parasitic nematodes are annually responsible for more than $100 billion crop yield loss worldwide and those considered as causing most of the damages are root-knot nematodes. These nematodes used to be controlled by chemicals that are now banned from use because of their poor specificity and high toxicity for the environment and human health. In the absence of sustainable alternative solutions, new control means, more specifically targeted against these nematodes and safe for the environment are needed. We searched in root-knot nematode genomes, genes conserved in various plant-damaging species while otherwise absent from the genomes of non target species such as those of chordates, plants, annelids, insect pollinators and mollusks. These genes are probably important for plant parasitism and their absence from non-target species make them interesting candidates for the development of more specific and safer control means. Further bioinformatics pruning of this set of genes yielded 16 novel candidates that could be biologically tested. Using RNA interference, we knocked down each of these 16 genes in a root-knot nematode and tested the effect on plant parasitism efficiency. Out of the 16 tested genes, 12 showed a significant and reproducible diminution of infestation when silenced and are thus particularly promising.

International audience

Considerable knowledge has been accumulated on the biochemical processes occurring during the ripening of cheese, which have in turn major consequences on flavour development . The flavour profiles of cheeses are complex and are variety specific . According to the ‘component balance’ theory, the flavour of cheese is the result of the correct balance of a wide variety of volatile flavour compounds originating from the degradation of milk constituents during ripening . Primary degradation of milk constituents leads to a whole range of flavour precursor compounds which are followed by a concerted series of secondary catabolic reactions that result in the formation of a variety of volatile compounds . Only some of the compounds formed directly contribute to cheese flavour . The formation of flavour compounds is mainly dependent on the enzymatic potentials of the microflora, their autolytic abilities and physiological states as well as on the manufacturing process, coagulant and indigenous milk enzymes . Some factors limiting the formation of flavour compounds have been recently identified . The availability of improved sensory and volatile instrumentation methodologies for the analysis of volatiles have aided our understanding and characterisation of cheese flavour. The analysis of the volatile profile of cheeses relies heavily on the extraction technique used (DHS, SHS, SPME, Thermal desorption, ITEX, HS-trap) as well as the sensitivity of the detectors . The use of GC-olfactometry to identify the odour activity of separated compounds has also served to improve our understanding of the contribution of individual compounds to the perception of flavour . However, to fully elucidate cheese flavour, descriptive sensory analysis should be conducted in tandem . New perspectives offered by analytical developments in -omic approaches can lead to a better knowledge of the potentials of micro-organisms (genome sequence), their global activity and physiology in cheese under starvation or at cold temperature (transcriptomics, flow cell cytometry, release of stress proteins observed by proteomics)

Considerable knowledge has been accumulated on the biochemical processes occurring during the ripening of cheese, which have in turn major consequences on flavour development . The flavour profiles of cheeses are complex and are variety specific . According to the ‘component balance’ theory, the flavour of cheese is the result of the correct balance of a wide variety of volatile flavour compounds originating from the degradation of milk constituents during ripening . Primary degradation of milk constituents leads to a whole range of flavour precursor compounds which are followed by a concerted series of secondary catabolic reactions that result in the formation of a variety of volatile compounds . Only some of the compounds formed directly contribute to cheese flavour . The formation of flavour compounds is mainly dependent on the enzymatic potentials of the microflora, their autolytic abilities and physiological states as well as on the manufacturing process, coagulant and indigenous milk enzymes . Some factors limiting the formation of flavour compounds have been recently identified . The availability of improved sensory and volatile instrumentation methodologies for the analysis of volatiles have aided our understanding and characterisation of cheese flavour. The analysis of the volatile profile of cheeses relies heavily on the extraction technique used (DHS, SHS, SPME, Thermal desorption, ITEX, HS-trap) as well as the sensitivity of the detectors . The use of GC-olfactometry to identify the odour activity of separated compounds has also served to improve our understanding of the contribution of individual compounds to the perception of flavour . However, to fully elucidate cheese flavour, descriptive sensory analysis should be conducted in tandem . New perspectives offered by analytical developments in -omic approaches can lead to a better knowledge of the potentials of micro-organisms (genome sequence), their global activity and physiology in cheese under starvation or at cold temperature (transcriptomics, flow cell cytometry, release of stress proteins observed by proteomics)

Background: Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. Symptoms observed in ewe S.aureus intramammary infections (IMI) are very different from one case to another: IMI ranges from subclinical mastitis to lethal gangrenous mastitis. Neither the S. aureus nor the host - factors contributing to the different outcomes are known. Objectives: Identify differences between S. aureus strains implied in the acuteness of IMI. Methods: Two S. aureus strains isolated from subclinical mastitis (O46) and gangrenous mastitis (O11) were compared by using PFGE, MLST, Micro - array, spa type, s as type, SNP, MLVA type, and RAPD. Proteomic profiles of these strains were also compared. After growth in iron depleted RPMI with low oxygen, conditions that mimic IMI context, cell wall, cytoplasmic and secreted proteins were extracted and ran on 2D gels . Results: O11 and O46 were genetically closely related. They had identical PFGE patterns (OV/OV', the main pattern in South East of France), were in the same MLST cluster. They shared 3615 identical targets using a pangenomic microarray. The core genes, the core variable genes, and the mobile genetic elements were essentially the same in both strains. The major differences were in spa type, sdrD , splE , fnbB , and the presence of the prophage genes (SAS0897, SAR1558, SAR0940, SAR2100, SACOL0343). RAPD show ed the presence of an unknown gene SAB 1979 in O11. Comparative proteomic revealed over secretion of some proteins ( hlg , isdB ,hly , lukF - PV, lukM, enolase) by O11. Conclusions: Absence of a prophage as well as surface protein variants were associated with enhanced virulence. Differences can either be explained by over expression of some virulence factors. This study shows that IMI acuteness can partly be explained by strain genetic content. The impact of host factors on ewe susceptibility to IMIs still remains to be determined.