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Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus–plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

SummaryClostridioides difficile infection (CDI) is an urgent threat in nosocomial infections, yet its associated microbiome remains poorly characterised. Among 534 metagenomes from 10 public CDI study populations we detected C. difficile in only 30% of samples, yet other toxigenic species with CDI-like symptomatology were prevalent, indicating possible CDI overdiagnosis. Tracking C. difficile across 42,900 metagenomes from 253 public studies, we found that prevalence, abundance and biotic context were age-dependent. C. difficile is a rare taxon associated with reduced diversity in healthy adults, but common and associated with increased diversity in infants. We identified a group of species co-occurring with C. difficile exclusively in healthy infants, enriched in obligate anaerobes and in species typical of the healthy adult gut microbiome. Overall, C. difficile in healthy infants is associated with multiple indicators of healthy gut microbiome maturation, suggesting that C. difficile is an important commensal in infants and that its asymptomatic carriage in adults depends on microbial context.

7 Pág., The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/., Amsterdam UMC; European Molecular Biology Laboratory (EMBL); Swiss National Science Foundation (SNSF) [205321_184955 to S.S.]; NCCR Microbiomes [51NF40_180575 to S.S. and C.v.M.]; German Federal Ministry of Education and Research (BMBF); de.NBI network [031A537B to P.B., 031L0181A to G.Z.]; German Research Foundation (DFG) [395357507 – SFB 1371 to G.Z., ‘NFDI4Microbiota’ to P.B.]; European Grant with code [PGC2018-098073-A-I00MCIU/AEI/FEDER to J.H.-C.]; Spanish Ministry of Universities [FPU-19/06635 to C.S.-L.]. Funding for open access charge: EMBL.

Fungal interactions with plant roots, either beneficial or detrimental, have a major impact on agriculture and ecosystems. The soil inhabiting ascomyceteFusarium oxysporum(Fo) constitutes a species complex of worldwide distribution causing vascular wilt in more than a hundred different crops. Individual isolates of the fungus exhibit host-specific pathogenicity, determined by proteinaceous effectors termed secreted in xylem (SIX). However, such isolates can also colonize roots of non-host plants asymptomatically as endophytes, or even protect them against pathogenic isolates. The molecular determinants of multi-host plant colonization are currently unknown. Here, we identified a set of fungal effectors termed ERCs (EarlyRootCompatibility effectors), which are secreted during early biotrophic growth of Fo on both host and non-host plants. In contrast to the strain-specific SIX effectors, which are encoded on accessory genomic regions, ERCs are encoded on core regions and are found across the entire Fo species complex as well as in other phytopathogens, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence on the host plant and rapid activation of plant immune responses, while in a non-pathogenic isolate it led to impaired root colonization and loss of biocontrol ability. Strikingly, some ERCs also contribute to Fo infection on the non-vascular land plantMarchantia polymorpha. Our results reveal an evolutionarily conserved mechanism for multi-host colonization by root infecting fungi.

Albugo candida is an obligate oomycete pathogen that infects many plants in the Brassicaceae family. We re-sequenced the genome of isolate Ac2V using PacBio long reads and constructed an assembly augmented by Illumina reads. The Ac2VPB genome assembly is 10% larger and more contiguous compared to a previous version. Our annotation of the new assembly, aided by RNASeq information, revealed a dramatic 250% expansion (40 to 110) in the CHxC effector class, which we redefined as “CCG” based on motif analysis. This class of effectors consist of arrays of phylogenetically related paralogs residing in gene sparse regions, and shows signatures of positive selection and presence/absence polymorphism. This work provides a resource that allows the dissection of the genomic components underlying A. candida adaptation and particularly the role of CCG effectors in virulence and avirulence on different hosts.

Advances in genome sequencing and assembly technologies are generating many high quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimised data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents more than 78% of the genome with a scaffold N50 of 88.8kbp that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNAseq and PacBio full-length cDNAs to identify 104,091 high confidence protein-coding genes and 10,156 non-coding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop. [Supplemental material is available for this article.]

Salmonellosis is an endemic human infection, associated with both sporadic cases and outbreaks throughout Colombia. Typhimurium is the most common Colombian serovar ofSalmonella enterica, responsible for 32.5% of theSalmonellainfections. Whole genome sequencing (WGS) is being used increasingly in Europe and the USA to study the epidemiology ofSalmonella, but there has not yet been a WGS-based analysis ofSalmonellaassociated with bloodstream infection in Colombia. Here, we analysed 209 genome sequences of ColombianS. Typhimurium and monophasicS. 4,[5],12:i:-isolates from Colombia from 1999 to 2017. We used a core genome-based maximum likelihood tree to define seven distinct clusters which were predominantly Sequence Type (ST) 19 isolates. We also identified the first ST313 and monophasic ST34 isolates to be reported in Colombia. The history of each cluster was reconstructed with a Bayesian tree to reveal a timeline of evolution. Cluster 7 was closely related to European multidrug-resistant (MDR) DT104. Cluster 4 became the dominant variant ofSalmonellain 2016, and resistance to nalidixic acid was associated with a plasmid-encodedqnrB19gene. Our findings suggest multiple transfers ofS. Typhimurium between Europe and Colombia.Author summaryThe large-scale genome sequencing ofSalmonellaTyphimurium and monophasicSalmonella4,[5],12:i:-involved bloodstream isolates from Colombia. The two serovars were responsible for about 1/3 ofSalmonellainfections in Colombia in the past 20 years. To identify the population structure we used Whole Genome Sequencing, performedin silicosequence typing, obtained phylogenetic trees, inferred the evolutionary history, detected the plasmids and prophages, and associated the antibiotic resistance (AMR) genotype with phenotype. Different clusters showed temporal replacement. The Colombian sequence type 313 was distinct from African lineages due to the absence of a key virulence-related gene,bstA. One of the Colombian clusters is likely to belong to the global epidemic of DT104, according to the evolutionary history and the AMR profile. The most common cluster in recent years was resistant to nalidixic acid and carried a plasmid-mediated antibiotic resistant geneqnrB19. Our findings will inform the ongoing efforts to combat Salmonellosis by Colombian public health departments.

Plant innate immunity relies on NLR receptors that recognize pathogen derived molecules and activate downstream signalling pathways. We analyzed the variation in copy number of NLR genes across flowering plants, and identified a number of species with a low number of NLRs relative to sister species. Two distinct lineages, one monocot (Lentibulariaceae) and one dicot (Alismatales) encapsulate four species with particularly few NLR genes. In these lineages, loss of NLRs coincided with loss of the well-known downstream immune signalling complex (EDS1-PAD4). When we expanded our analysis across the whole proteomes, we were able to identify other characterized immune genes absent only in Lentibulariaceae and Alismatales. Additionally, we identified a small subset of genes with unknown function convergently lost in all four species. We predicted that some of these genes may have a role in plant immunity. Gene expression analyses confirmed that a group of these genes was differentially expressed under pathogen infection. Another subset of these genes was differentially expressed upon drought providing further evidence of a link between the drought and plant immunity.

Background The plant immune system is innate and encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes. Results We show that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncover a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events reveals that homologous receptors can be fused to diverse domains. Furthermore, we discover a 43 amino acid long motif associated with this dominant integration clade which is located immediately upstream of the fusion site. Sequence analysis reveals that DNA transposition and/or ectopic recombination are the most likely mechanisms of formation for nucleotide binding leucine rich repeat proteins with integrated domains. Conclusions The identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen “baits.” Electronic supplementary material The online version of this article (10.1186/s13059-018-1392-6) contains supplementary material, which is available to authorized users.

Nature Plants 1, 15025 (2015); published online 23 March 2015; corrected 28 May 2015 In Figure 3e of the version of this Article originally published, the wt/wt control graft and ck1.2/ck1.2 mutant control graft were mislabelled. This error has been corrected.

International audience; Directed plant cell growth is governed by deposition and alterations of cell wall components under turgor pressure. A key regulatory element of anisotropic growth, and hence cell shape, is the directional deposition of cellulose microfibrils. The microfibrils are synthesized by plasma membrane-located cellulose synthase complexes that co-align with and move along cortical microtubules. That the parallel relation between cortical microtubules and extracellular microfibrils is causal has been named the alignment hypothesis. Three recent studies revealed that the previously identified pom2 mutant codes for a large cellulose synthases interacting (CSI1) protein which also binds cortical microtubules. This review summarizes these findings, provides structure function models and discusses the inferred mechanisms in the context of plant growth.

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by < 25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

To cope with the ever-increasing amount of sequence data generated in the field of genomics, the demand for efficient and fast database searches that drive functional and structural annotation in both large- and small-scale genome projects is on the rise. The tools of the BLAST+ suite are the most widely employed bioinformatic method for these database searches. Recent trends in bioinformatics application development show an increasing number of JavaScript apps that are based on modern frameworks such as Node.js. Until now, there is no way of using database searches with the BLAST+ suite from a Node.js codebase. We developed blastjs, a Node.js library that wraps the search tools of the BLAST+ suite and thus allows to easily add significant functionality to any Node.js-based application. blastjs is a library that allows the incorporation of BLAST+ functionality into bioinformatics applications based on JavaScript and Node.js. The library was designed to be as user-friendly as possible and therefore requires only a minimal amount of code in the client application. The library is freely available under the MIT license at https://github.com/teammaclean/blastjs .

Background The genus Cuscuta is a group of parasitic plants that are distributed world-wide. The process of parasitization starts with a Cuscuta plant coiling around the host stem. The parasite’s haustorial organs then establish a vascular connection allowing for access to the phloem content. The host and the parasite form new cellular connections, suggesting coordination of developmental and biochemical processes. Simultaneous monitoring of gene expression in the parasite’s and host’s tissues may shed light on the complex events occurring between the parasitic and host cells and may help to overcome experimental limitations (i.e. how to separate host tissue from Cuscuta tissue at the haustorial connection). A novel approach is to use bioinformatic analysis to classify sequencing reads as either belonging to the host or to the parasite and to characterize the expression patterns. Owing to the lack of a comprehensive genomic dataset from Cuscuta spp., such a classification has not been performed previously. Results We first classified RNA-Seq reads from an interface region between the non-model parasitic plant Cuscuta japonica and the non-model host plant Impatiens balsamina. Without established reference sequences, we classified reads as originating from either of the plants by stepwise similarity search against de novo assembled transcript sets of C. japonica and I. balsamina, unigene sets of the same genus, and cDNA sequences of the same family. We then assembled de novo transcriptomes from the classified read sets. We assessed the quality of the classification by mapping reads to contigs of both plants, achieving a misclassification rate low enough (0.22-0.39%) to be used reliably for differential gene expression analysis. Finally, we applied our read classification method to RNA-Seq data from the interface between the non-model parasitic plant C. japonica and the model host plant Glycine max. Analysis of gene expression profiles at 5 parasitizing stages revealed differentially expressed genes from both C. japonica and G. max, and uncovered the coordination of cellular processes between the two plants. Conclusions We demonstrated that reliable identification of differentially expressed transcripts in undissected interface region of the parasite-host association is feasible and informative with respect to differential-expression patterns. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0066-6) contains supplementary material, which is available to authorized users.

The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants? Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.

Climate models predict an increased likelihood of seasonal droughts for many areas of the world. Breeding for drought tolerance could be accelerated by marker-assisted selection. As a basis for marker identification, we studied the genetic variance, predictability of field performance and potential costs of tolerance in potato (Solanum tuberosum L.). Potato produces high calories per unit of water invested, but is drought-sensitive. In 14 independent pot or field trials, 34 potato cultivars were grown under optimal and reduced water supply to determine starch yield. In an artificial dataset, we tested several stress indices for their power to distinguish tolerant and sensitive genotypes independent of their yield potential. We identified the deviation of relative starch yield from the experimental median (DRYM) as the most efficient index. DRYM corresponded qualitatively to the partial least square model-based metric of drought stress tolerance in a stress effect model. The DRYM identified significant tolerance variation in the European potato cultivar population to allow tolerance breeding and marker identification. Tolerance results from pot trials correlated with those from field trials but predicted field performance worse than field growth parameters. Drought tolerance correlated negatively with yield under optimal conditions in the field. The distribution of yield data versus DRYM indicated that tolerance can be combined with average yield potentials, thus circumventing potential yield penalties in tolerance breeding.

Molecular biomarkers are molecules whose concentrations in a biological system inform about the current phenotypical state and, more importantly, may also be predictive of future phenotypic trait endpoints. The identification of biomarkers has gained much attention in targeted plant breeding since technologies have become available that measure many molecules across different levels of molecular organization and at decreasing costs. In this chapter, we outline the general strategy and workflow of conducting biomarker discovery studies. Critical aspects of study design as well as the statistical data analysis and model building will be highlighted.

In plant breeding, plants have to be characterised precisely, consistently and rapidly by different people at several field sites within defined time spans. For a meaningful data evaluation and statistical analysis, standardised data storage is required. Data access must be provided on a long-term basis and be independent of organisational barriers without endangering data integrity or intellectual property rights. We discuss the associated technical challenges and demonstrate adequate solutions exemplified in a data management pipeline for a project to identify markers for drought tolerance in potato. This project involves 11 groups from academia and breeding companies, 11 sites and four analytical platforms. Our data warehouse concept combines central data storage in databases and a file server and integrates existing and specialised database solutions for particular data types with new, project-specific databases. The strict use of controlled vocabularies and the application of web-access technologies proved vital to the successful data exchange between diverse institutes and data management concepts and infrastructures. By presenting our data management system and making the software available, we aim to support related phenotyping projects.

Molecular biomarkers are molecules whose concentrations in a biological system inform about the current phenotypical state and, more importantly, may also be predictive of future phenotypic trait endpoints. The identification of biomarkers has gained much attention in targeted plant breeding since technologies have become available that measure many molecules across different levels of molecular organization and at decreasing costs. In this chapter, we outline the general strategy and workflow of conducting biomarker discovery studies. Critical aspects of study design as well as the statistical data analysis and model building will be highlighted.

RNA folding is assumed to be a hierarchical process. The secondary structure of an RNA molecule, signified by base-pairing and stacking interactions between the paired bases, is formed first. Subsequently, the RNA molecule adopts an energetically favorable three-dimensional conformation in the structural space determined mainly by the rotational degrees of freedom associated with the backbone of regions of unpaired nucleotides (loops). To what extent the backbone conformation of RNA loops also results from interactions within the local sequence context or rather follows global optimization constraints alone has not been addressed yet. Because the majority of base stacking interactions are exerted locally, a critical influence of local sequence on local structure appears plausible. Thus, local loop structure ought to be predictable, at least in part, from the local sequence context alone. To test this hypothesis, we used Random Forests on a nonredundant data set of unpaired nucleotides extracted from 97 X-ray structures from the Protein Data Bank (PDB) to predict discrete backbone angle conformations given by the discretized η/θ-pseudo-torsional space. Predictions on balanced sets with four to six conformational classes using local sequence information yielded average accuracies of up to 55%, thus significantly better than expected by chance (17%–25%). Bases close to the central nucleotide appear to be most tightly linked to its conformation. Our results suggest that RNA loop structure does not only depend on long-range base-pairing interactions; instead, it appears that local sequence context exerts a significant influence on the formation of the local loop structure.

International audience; ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING (Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from