SummaryCrop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild pathosystems, we carried out a multi-year, multi-site survey of Pseudomonas in its natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a ubiquitous pathogenic clade. Sequencing of 1,524 genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically identifiable pathogenic sublineages. There is differentiation at the level of both gene content and disease phenotype, although the differentiation may not provide fitness advantages to specific sublineages. The coexistence of sublineages indicates that in contrast to crop systems, no single strain has been able to overtake the studied A. thaliana populations in the recent past. Our results suggest that selective pressures acting on a plant pathogen in wild hosts are likely to be much more complex than those in agricultural systems.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

BackgroundA high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read–only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates.ResultsHere, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs.ConclusionsThe field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers.

BackgroundThe Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.ResultsSequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn’t generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.ConclusionThe described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3936-7) contains supplementary material, which is available to authorized users.

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.

A high quality genome sequence of your model organism is an essential starting point for many studies. Old clone based methods are slow and expensive, whereas faster, cheaper short read only assemblies can be incomplete and highly fragmented, which minimises their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, human. However, plant genomes can be much more repetitive and larger than human, and plant biology makes obtaining high quality DNA free from contaminants difficult. Reflecting their challenging nature we observe that plant genome assembly statistics are typically poorer than for vertebrates. Here we compare Illumina short read, PacBio long read, 10x Genomics linked reads, Dovetail Hi-C and BioNano Genomics optical maps, singly and combined, in producing high quality long range genome assemblies of the potato species S. verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA, compute requirements and sequencing costs. We expect our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers.

SummaryCrop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine 20 whether similar boom-and-bust scenarios hold for wild plant pathogens, we carried out a multi-year multi-site 21 survey of Pseudomonas in the natural host Arabidopsis thaliana. The most common Pseudomonas lineage 22 corresponded to a pathogenic clade present in all sites. Sequencing of 1,524 Pseudomonas genomes revealed 23 this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically distinct 24 pathogenic sublineages. These sublineages have expanded in parallel within the same populations and are 25 differentiated both at the level of gene content and disease phenotype. Such coexistence of diverse sublineages 26indicates that in contrast to crop systems, no single strain has been able to overtake these A. thaliana 27 populations in the recent past. Our results suggest that the selective pressures acting on a plant pathogen in 28 wild hosts may be more complex than those in agricultural systems. 29 Introduction 30In agricultural and clinical settings, pathogenic colonizations are frequently associated with expansions of single 31 or a few genetically identical microbial lineages (Butler et al., 2013; Cai et al., 2011;Kolmer, 2005; Park et al., 32 2015;Stukenbrock and McDonald, 2008; Yoshida et al., 2013). The conditions that lead to such epidemics-such 33 as reduced host genetic diversity (Zhu et al., 2000), absence of competing microbial communities (Brown et al., 34 2013) or high transmission rates (Park et al., 2015)-are, however, by no means a universal feature of 35 pathogenic infections. Instead, many, if not most, pathogens can colonize host populations that are both 36 genetically diverse and that can accommodate a diversity of other microbes (Barrett et al., 2009; Falkinham et 37 al., 2015;Woolhouse et al., 2001). 38Factors that drive pathogen success in such more complex situations are less well understood than for 39 clonal epidemics. For example, if a pathogen species persists at high numbers in non-host environments, does 40 each host become infected by a different pathogen strain? Or does a multitude of genetically distinct pathogens 41 infect each host? And do different colonizing strains use disparate mechanisms to become established even 42 within genetically similar host individuals? The answers to these questions inform on how (and if) a host 43 population can evolve partial or even complete pathogen resistance (Anderson and May, 1982; Barrett et al., 44 2009; Karasov et al., 2014a;Laine et al., 2011). Several studies over the past 20 years have attempted to infer 45 the distributions of non-epidemic pathogens in both host and non-host environments (Falkinham et al., 2015; 46 Wiehlmann et al., 2007). These studies, which have observed a range of different patterns, are unfortunately 47 often limited to the historic strains that are available, and the conclusions vary for different collections, even of 48 the same pathogen ...

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca2+]i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.