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JvS, MH and SvdE were supported by a grant from the Applied and Technical Science domain (TTW) of the Netherlands Organization for Scientific Research (NWO) under grant no. 14708. PT received support from the University of St Andrews Bioinformatics Unit (AMD3BIOINF), funded by Wellcome Trust ISSF award 105621/Z/14/Z. MS benefitted from funding by a VENI grant (17282) from the NWO domain Applied and Engineering Sciences. Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression ('DOG boxes') were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion. Publisher PDF

Understanding the phylogeographic history of a group and identifying the factors contributing to speciation is an important challenge in evolutionary biology. Vicariance has clearly played a major role in diversification across diverse groups of organisms, but how this interacts with intrinsic biological features such as changes in reproductive system or sexual selection is less well understood. TheGoodeinaeare a Mexican endemic group of live-bearing fish. Here, we develop genomic resources for species within theGoodeinaeand use phylogenomic approaches to characterise their evolutionary history. We sequenced, assembled and annotated the genomes of four newGoodeinaegenomes, includingA. toweri, the only matrotrophic live-bearing fish without a trophotaenia in the group. We produced a phylogeny of theGoodeinaeand estimate timings of species divergence. We determined the extent and timing of introgression between the species to assess if this may have occurred during an early radiation, or in more recent episodes of secondary contact. We also analyse patterns in the changes of effective population size for the species and examine the time course of expansion and decline. We used branch-site models to detect genome-wide positive selection acrossGoodeinae, and we specifically ask if this differs inA. toweri, where reversal of placental viviparity has recently occurred. We find that Goodeinae diverged rapidly, with reductions in effective population size after each split. Contrary to expectations, we find evidence of gene flow between geographically isolated species, suggesting vicariant speciation was supplemented by limited post-speciation gene flow, potentially as a result of river piracy. Genes under positive selection in the group are likely to be associated with the switch to live-bearing. Overall, our studies suggest that both volcanism driven vicariance and changes in reproductive mode influenced radiation in the Goodeinae.

The transition from oviparity to viviparity has occurred independently over a hundred times across vertebrates, presenting a compelling case of phenotypic convergence. However, whether repeated, independent evolution of viviparity is driven by redeployment of similar genetic mechanisms and whether these leave a common genetic signature in genomic divergence remains unknown. Whilst investigations into the evolution of viviparity have demonstrated striking similarity among the genes and pathways involved across vertebrate groups, quantitative tests for genome-wide convergence provide ambivalent answers. Here, we investigate molecular convergence during independent transitions to viviparity across an order of ray-finned freshwater fish (Cyprinodontiformes). We assembled de novo and publicly-available genomes of viviparous and oviparous species to quantify molecular convergence across coding and non-coding regions. We found no evidence for an excess of molecular convergence in amino acid substitutions and rates of sequence divergence, implying independent genetic changes are associated with these transitions. However, statistical power and biological confounds (hemiplasy and introgression) could constrain our ability to detect correlated evolution. We therefore also identified candidate genes with potential signatures of molecular convergence in viviparous Cyprinodontiformes lineages. While we detected no evidence of positive or relaxed selection for these genes in branches associated with the evolution of viviparity in Cyprinodontiformes, motif-enrichment and gene ontology analyses suggest transcriptional changes associated with early morphogenesis, brain development and immunity occurred alongside the evolution of viviparity. Overall, our findings indicate that an excess of molecular convergence, at any level, is not strongly associated with independent transitions to viviparity in these fish.

Funding: Breast Cancer Now (Grant Number(s): 2018JulPR1086), Wellcome Trust (GrantNumber(s): 105621/Z/14/Z), Melville Charitable Trust. The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilising HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T cell responses in previous studies. Our data thus shows the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV. Publisher PDF

Plasmodium knowlesi, a malaria parasite of old-world macaque monkeys, is used extensively to model Plasmodium biology. Recently P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes un-complicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum.As such P. knowlesi presents a unique opportunity to inform an experimental model for malaria with clinical data from same-species human infections.Experimental lines of P. knowlesi represent well characterised genetically static parasites and to maximise their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization.The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence P. knowlesi parasites from archived clinical samples. The sequencing platform and assembly pipeline was designed to facilitate capturing data on important multiple gene families, including the P. knowlesi schizont-infected cell agglutination (SICA) var genes and the Knowlesi-Interspersed Repeats (KIR) genes.The SICAvar and KIR gene families code for antigenically variant proteins that have been difficult to resolve and characterise. Analyses presented here suggest that the family members have arisen through a process of gene duplication, selection pressure and variation. Highly evolving genes tend to be located proximal to genetic elements that drive change rather than regions that support core gene conservation. For example, the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) gene family members are restricted to relatively unstable sub-telomeric regions. In contrast the SICAvar and KIR genes are located throughout the genome but as the study presented here shows, they occupy otherwise gene-sparse chromosomal locations.The novel methods presented here offer the malaria research community new tools to generate comprehensive genome sequence data from small clinical samples and renewed insight into these complex real-world parasites.Author summaryMalaria is a potentially severe disease caused by parasite species within genus Plasmodium. Even though the number of cases is in decline there were over 200 million reported cases of malaria in 2019 that resulted in >400,000 deaths. Despite huge research efforts we still do not understand precisely how malaria makes some individuals very ill and by extension how to successfully augment and manage severe disease.Here we developed a novel method to generate comprehensive robust genome sequences from the malaria parasite Plasmodium knowlesi collected from clinical samples.We propose to use the method and initial data generated here to begin to build a resource to identify disease associated genetic traits of P. knowlesi taken from patient’s samples. In addition to the methodology, what further sets this work apart is the unique opportunity to utilize same-species experimental P. knowlesi parasites to discover a potential role for particular parasite traits in the differential disease progression we observe in patients with P. knowlesi malaria.While we developed the methods to study severe malaria, they are affordable and accessible, and offer the wider malaria research community the means to add context and insight into real-world malaria parasites.

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (‘DOG boxes’) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

Left-right asymmetries in the nervous system (lateralisation) influence a broad range of behaviours, from social responses to navigation and language. The role and pathways of endogenous and environmental mechanisms in the ontogeny of lateralisation remains to be established. The domestic chick is a model of both endogenous and experience-induced lateralisation driven by light exposure. Following the endogenous rightward rotation of the embryo, the asymmetrical position in the egg results in a greater exposure of the right eye to environmental light. To identify the genetic pathways activated by asymmetric light stimulation, and their time course, we exposed embryos to different light regimes: darkness, 6 hours of light and 24 hours of light. We used RNA-seq to compare gene expression in the right and left retinas and telencephalon. As expected, no differential expression between left and right was present in darkness. We detected differential gene expression in right vs left retina after 6 hours of light exposure. This difference disappeared before 24 hours of light exposure, suggesting that light-induced activation is a self-terminating phenomenon. This transient effect of light exposure was associated with a downregulation of the sensitive-period mediator gene DIO2 (iodothyronine deiodinase 2) in the right retina. No differences between genes expressed in the right vs. left telencephalon were detected. Gene networks associated with lateralisation were connected to vascularisation, cell motility, and the extracellular matrix. Interestingly, we know that the extracellular matrix– including the differentially expressed PDGFRB (platelet-derived growth factor receptor β) gene – is involved in both sensitive periods and in the endogenous chiral mechanism of primary cilia, that drives lateralisation. Our data show a similarity between endogenous and experience-driven lateralisation, identifying functional gene networks that affect lateralisation in a specific time window.

Plant-parasitic nematodes are a major, and in some cases a dominant, threat to crop production in all agricultural systems. The relative scarcity of classical resistance genes highlights a pressing need to identify new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major stages of the interaction. This novel approach enabled the analysis of the hologenome of the infection site, to identify metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that the highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is critically important for parasitism. Knockout of either the plant-encoded or the now nematode-encoded steps in the pathway blocks parasitism. Our experiments establish a reference for cyst nematodes, use this platform to further our fundamental understanding of the evolution of plant-parasitism by nematodes, and show that understanding congruent differential expression of metabolic pathways represents a new way to find nematode susceptibility genes, and thereby, targets for future genome editing-mediated generation of nematode-resistant crops.

Funding: S.C.W., L.R.J.W., P.H., and J.M. were supported by funding from the Scottish Government Rural and Environment Science and Analytical Services Division (RESAS). P.T. and bioinformatics and computational biology analyses were supported by the University of St Andrews Bioinformatics Unit (AMD3BIOINF), funded by Wellcome Trust ISSF award 105621/Z/14/Z. R.R.V. was funded by the Swedish Research Council Formas (grant 2019-01316) and Helge Ax:son Johnsons foundation. Species of Phytophthora, plant pathogenic eukaryotic microbes, can cause disease on many tree species. Genome sequencing of species from this genus has helped to determine components of their pathogenicity arsenal. Here, we sequenced genomes for two widely distributed species, Phytophthora pseudosyringae and Phytophthora boehmeriae, yielding genome assemblies of 49 and 40 Mb, respectively. We identified more than 270 candidate disease promoting RXLR effector coding genes for each species, and hundreds of genes encoding candidate plant cell wall degrading carbohydrate active enzymes (CAZymes). These data boost genome sequence representation across the Phytophthora genus, and form resources for further study of Phytophthora pathogenesis. Publisher PDF

Root-knot nematodes (genus Meloidogyne) are the major contributor to crop losses caused by nematodes. These nematodes secrete effector proteins into the plant, derived from two sets of pharyngeal gland cells, to manipulate host physiology and immunity. Successful completion of the life cycle, involving successive molts from egg to adult, covers morphologically and functionally distinct stages and will require precise control of gene expression, including effector genes. The details of how root-knot nematodes regulate transcription remain sparse. Here, we report a life stage-specific transcriptome of Meloidogyne incognita. Combined with an available annotated genome, we explore the spatio-temporal regulation of gene expression. We reveal gene expression clusters and predicted functions that accompany the major developmental transitions. Focusing on effectors, we identify a putative cis-regulatory motif associated with expression in the dorsal glands, providing an insight into effector regulation. We combine the presence of this motif with several other criteria to predict a novel set of putative dorsal gland effectors. Finally, we show this motif, and thereby its utility, is broadly conserved across the Meloidogyne genus, and we name it Mel-DOG. Taken together, we provide the first genome-wide analysis of spatio-temporal gene expression in a root-knot nematode and identify a new set of candidate effector genes that will guide future functional analyses.

Species of Phytophthora, plant pathogenic eukaryotic microbes, can cause disease on many tree species. Genome sequencing of species from this genus has helped to determine components of their pathogenicity arsenal. Here we sequenced and assembled genomes for three widely distributed species, Phytophthora gonapodyides, P. pseudosyringae and P. boehmeriae. The genome assemblies ranged from 40 Mb to 96 Mb. We identified more than 250 candidate disease promoting RXLR effector coding genes for each species, and hundreds of genes encoding candidate plant cell wall degrading carbohydrate active enzymes (CAZymes). These data boost genome sequence representation across the Phytophthora genus, within species, and form resources for further study of Phytophthora pathogenesis.

The potato cyst nematode Globodera pallida acquires all of its nutrients from an elaborate feeding site that it establishes in a host plant root. Normal development of the root cells is re-programmed in a process coordinated by secreted nematode effector proteins. The biological function of the G. pallida GpIA7 effector was investigated in this study. GpIA7 is specifically expressed in the subventral pharyngeal glands of pre-parasitic stage nematodes. Ectopic expression of GpIA7 in potato plants affected plant growth and development, suggesting a potential role for this effector in feeding site establishment. Potato plants overexpressing GpIA7 were shorter, with reduced tuber weight and delayed flowering. We provide evidence that GpIA7 associates with the plant growth regulator StEBP1 (ErbB-3 epidermal growth factor receptor-binding protein 1). GpIA7 modulates the regulatory function of StEBP1, altering the expression level of downstream target genes, including ribonucleotide reductase 2, cyclin D3;1, and retinoblastoma related 1, which are down-regulated in plants overexpressing GpIA7. We provide an insight into the molecular mechanism used by the nematode to manipulate the host cell cycle and demonstrate that this may rely, at least in part, on hindering the function of host EBP1., We report the first secreted nematode effector protein that can alter the expression of key host cell cycle components by modulating the regulatory function of a plant growth regulator.

This work was funded through an AHDB PhD award, the USDA GLOBAL Project and by the Rural and Environmental Science and Analytical Services division of the Scottish Government. Bioinformatics and computational analyses for the generation of the new G. pallida genome assembly were supported by the University of St Andrews Bioinformatics Unit, which is funded by a Wellcome Trust ISSF award (grant 105621/Z/14/Z). Although the use of natural resistance is the most effective management approach against the potato cyst nematode (PCN) Globodera pallida, the existence of pathotypes with different virulence characteristics constitutes a constraint towards this goal. Two resistance sources, GpaV (from Solanum vernei) and H3 from S. tuberosum ssp. andigena CPC2802 (from the Commonwealth Potato Collection) are widely used in potato breeding programmes in European potato industry. However, the use of resistant cultivars may drive strong selection towards virulence, which allows the increase in frequency of virulent alleles in the population and therefore, the emergence of highly virulent nematode lineages. This study aimed to identify Avirulence (Avr) genes in G. pallida populations selected for virulence on the above resistance sources, and the genomic impact of selection processes on the nematode. The selection drive in the populations was found to be specific to their genetic background. At the genomic level, 11 genes were found that represent candidate Avr genes. Most of the variant calls determining selection were associated with H3-selected populations, while many of them seem to be organised in genomic islands facilitating selection evolution. These phenotypic and genomic findings combined with histological studies performed revealed potential mechanisms underlying selection in G. pallida. Publisher PDF

Somnath Pokhare was supported by a fellowship provided by the Indian Council of Agricultural Research (ICAR), Government of India. This work was also supported by Royal Society International Exchanges award number IE150670 and by the Rural and Environmental Science and Analytical Services Division of the Scottish Government. SEvdA is supported by Biotechnology and Biological Sciences Research Council grants BB/R011311/1 and BB/S006397/1. This work benefited from interactions funded through COST Action FA1208. The authors thank Dr Danny Coyne (IITA) for providing the starter culture of the H. sacchari population used for this work. Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit which is funded by a Wellcome Trust ISSF award [grant 105621/Z/14/Z]. Interactions between plant‐parasitic nematodes and their hosts are mediated by effectors, i.e. secreted proteins that manipulate the plant to the benefit of the pathogen. To understand the role of effectors in host adaptation in nematodes, we analysed the transcriptome of Heterodera sacchari , a cyst nematode parasite of rice (Oryza sativa ) and sugarcane (Saccharum officinarum ). A multi‐gene phylogenetic analysis showed that H. sacchari and the cereal cyst nematode Heterodera avenae share a common evolutionary origin and that they evolved to parasitise monocot plants from a common dicot‐parasitic ancestor. We compared the effector repertoires of H. sacchari with those of the dicot parasites Heterodera glycines and Globodera rostochiensis to understand the consequences of this transition. While, in general, effector repertoires are similar between the species, comparing effectors and non‐effectors of H. sacchari and G. rostochiensis shows that effectors have accumulated more mutations than non‐effectors. Although most effectors show conserved spatiotemporal expression profiles and likely function, some H. sacchari effectors are adapted to monocots. This is exemplified by the plant‐peptide hormone mimics, the CLAVATA3/EMBRYO SURROUNDING REGION‐like (CLE) effectors. Peptide hormones encoded by H. sacchari CLE effectors are more similar to those from rice than those from other plants, or those from other plant‐parasitic nematodes. We experimentally validated the functional significance of these observations by demonstrating that CLE peptides encoded by H. sacchari induce a short root phenotype in rice, whereas those from a related dicot parasite do not. These data provide a functional example of effector evolution that co‐occurred with the transition from a dicot‐parasitic to a monocot‐parasitic lifestyle. Publisher PDF

The diversity of Phytophthora species in soils collected from 14 highly disturbed sites in northern Britain, including botanic gardens, arboreta, public parks and other amenity woodland sites, was analysed using a molecular technique known as DNA metabarcoding. This technique enables the identification of multiple species present in a single environmental sample based on a DNA ‘barcode’ unique to each species. The genus Phytophthora was targeted in this study due to its increasing impact on Britain’s forests and woodlands over thelast 20 years. The introduction and spread of new Phytophthora species into Britain has been strongly associated with the movement of traded containerised plants, with a number of Phytophthora outbreaks reported on host trees located in public gardens and parks that had recently undergone planting or landscape regeneration schemes. This study was undertaken to assess the extent to which these highly disturbed sites with extensive planting regimes act as harbours for woody-host infecting Phytophthora species. A total of 23 Phytophthora species, the majority of which are known to be pathogens of woody hosts, were detected across the 14 sites sampled. These included four quarantine-regulated pathogens and four species notpreviously recorded in Britain. Also detected were three as-yet undescribed Phytophthora species and nine oomycete sequences with no clear match to any known genus. There was no effect of geographical location, elevation, underlying soil type, host family or host health status on the Phytophthora assemblages at each site, suggesting that the Phytophthora communities detected are likely to comprise introduced species associated with planting programmes. P. austrocedri and P. pseudosyringae were two of the most abundant Phytophthoraspecies detected, both of which cause serious damage to trees and are regarded as fairly recent introductions to Britain. The practical implications of the findings in terms of mitigating Phytophthora introduction, spread and impact at botanic gardens, arboreta and urban parks are discussed.

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans. Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection., The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.

This work was supported by the Biotechnology and Biological Sciences Research Council (BB/M014207/1 to SEvdA), European Research Council (310190- APHIDHOST to JIBB), and Royal Society of Edinburgh (fellowship to JIBB). Aphids are a diverse group of taxa that contain agronomically important species, which vary in their host range and ability to infest crop plants. The genome evolution underlying agriculturally important aphid traits is not well understood. We generated draft genome assemblies for two aphid species: Myzus cerasi (black cherry aphid), and the cereal specialist Rhopalosiphum padi. Using a de novo gene prediction pipeline on both these, and three additional aphid genome assemblies (Acyrthosiphon pisum, D. noxia and M. persicae), we show that aphid genomes consistently encode similar gene numbers. We compare gene content, gene duplication, synteny, and putative effector repertoires between these five species to understand the genome evolution of globally important plant parasites. Aphid genomes show signs of relatively distant gene duplication, and substantial, relatively recent, gene birth. Putative effector repertoires, originating from duplicated and other loci have an unusual genomic organisation and evolutionary history. We identify a highly conserved effector-pair that is tightly physically-linked in the genomes of all aphid species tested. In R. padi, this effector pair is tightly transcriptionally-linked, and shares an unknown transcriptional control mechanism with a subset of approximately 50 other putative effectors and secretory proteins. This study extends our current knowledge on the evolution of aphid genomes and reveals evidence for an as of yet unknown shared control mechanism, which underlies effector expression, and ultimately plant parasitism. Publisher PDF

Result files for the analysis generated during: Transcriptome analysis of the sugarcane cyst nematode Heterodera sacchari shows signatures of adaptation to a monocot host

Aphids are phloem-feeding insects that cause yield losses to crops globally. These insects feature complex life cycles, which in the case of many agriculturally important species involves the use of primary and secondary host plant species. Whilst host alternation between primary and secondary host can occur in the field depending on host availability and the environment, aphid populations maintained as laboratory stocks generally are kept under conditions that allow asexual reproduction by parthenogenesis on secondary hosts. Here, we usedMyzus cerasi(black cherry aphid) to assess aphid transcriptional differences between populations collected from primary hosts in the field and those adapted to secondary hosts under controlled environment conditions. Adaptation experiments ofM. cerasicollected from local cherry tress to reported secondary host species resulted in low survival rates. Moreover, aphids were unable to survive on secondary host Land cress, unless first adapted to another secondary host, cleavers. Transcriptome analyses of populations collected from primary host cherry in the field and the two secondary host plant species in a controlled environment showed extensive transcriptional plasticity to a change in host environment, with predominantly genes involved in redox reactions differentially regulated. Most of the differentially expressed genes across theM. cerasipopulations from the different host environments were duplicated and we found evidence for differential exon usage. In contrast, we observed only limited transcriptional to a change in secondary host plant species.

Analysis of the genome sequence of the potato cyst nematode, Globodera pallida, has shown that a substantial gene family (approximately 300 sequences) of proteins containing a SPRY domain is present in this species. This is a huge expansion of the gene family as compared to other organisms, including other plant-parasitic nematodes. Some SPRY domain proteins from G. pallida and G. rostochiensis have signal peptides for secretion and are deployed as effectors. One of these SPRYSEC proteins has been shown to suppress host defence responses. We describe further analysis of this gene family in G. pallida. We show that only a minority (10%) of the SPRY domain proteins in this species have a predicted signal peptide for secretion and that the presence of a signal peptide is strongly correlated with the corresponding gene being expressed at the early stages of parasitism. The data suggest that while the gene family is greatly expanded, only a minority of SPRY domain proteins in G. pallida are SPRYSEC candidate effectors. We show that several new SPRYSECs from G. pallida are expressed in the dorsal gland cell and demonstrate that some, but not all, of the SPRYSECs can suppress the hypersensitive response induced by co-expression of the resistance gene Gpa2 and its cognate avirulence factor RBP-1 in Nicotiana benthamiana.

Work described in this article was partly funded by the Rural & Environment Science & Analytical Services Division of the Scottish Government and by two grants from the French National Research Agency (ANR) under the Genoplante program (project ANR-PCS-08-GENO-166 NEMATARGETS) and the JCJC program (project ANR-13-JSV7-0006—ASEXEVOL). This work was facilitated by interactions funded through COST action FA1208 and Royal Society International Exchange award IE130707. Sebastian Eves-van den Akker is supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/M014207/1. Nematodes have evolved the ability to parasitize plants on at least four independent occasions, with plant parasites present in Clades 1, 2, 10 and 12 of the phylum. In the case of Clades 10 and 12, horizontal gene transfer of plant cell wall degrading enzymes from bacteria and fungi has been implicated in the evolution of plant parasitism. We have used ribonucleic acid sequencing (RNAseq) to generate reference transcriptomes for two economically important nematode species, Xiphinema index and Longidorus elongatus, representative of two genera within the early-branching Clade 2 of the phylum Nematoda. We used a transcriptome-wide analysis to identify putative horizontal gene transfer events. This represents the first in-depth transcriptome analysis from any plant-parasitic nematode of this clade. For each species, we assembled ~30 million Illumina reads into a reference transcriptome. We identified 62 and 104 transcripts, from X. index and L. elongatus, respectively, that were putatively acquired via horizontal gene transfer. By cross-referencing horizontal gene transfer prediction with a phylum-wide analysis of Pfam domains, we identified Clade 2-specific events. Of these, a GH12 cellulase from X. index was analysed phylogenetically and biochemically, revealing a likely bacterial origin and canonical enzymatic function. Horizontal gene transfer was previously shown to be a phenomenon that has contributed to the evolution of plant parasitism among nematodes. Our findings underline the importance and the extensiveness of this phenomenon in the evolution of plant-parasitic life styles in this speciose and widespread animal phylum. Publisher PDF

The James Hutton Institute receives funding from the Scottish Government. This work benefited from interactions funded through COST FA1208. There has been a huge amount of work put into identifying and characterising effectors from plant-parasitic nematodes in recent years. Although this work has provided insights into the mechanisms by which nematodes can infect plants, the potential translational outputs of much of this research are not always clear. This short article will summarise how developments in effector biology have allowed, or will allow, new control strategies to be developed, drawing on examples from nematology and from other pathosystems. Postprint

There is no current consensus on what constitutes the ideal management of emphysematous pyelonephritis. We review the current management strategies including the role of nephron preserving percutaneous drainage in the treatment of emphysematous pyelonephritis.We searched MEDLINE, PubMed, EMBASE, CINAHL and the Cochrane Library from 1966 to 2006. Abstracts were reviewed including all types of studies from prospective randomized controlled studies to small retrospective series. All relevant English language articles reporting on at least 5 patients were reviewed.Ten retrospective studies on 210 patients with emphysematous pyelonephritis met the inclusion criteria. There were 167 females and 43 males with a mean age of 57 years (range 24 to 83). Of the patients 96% had diabetes mellitus and 29% had urinary tract obstruction. The diagnostic accuracy of computerized tomography was 100%. Escherichia coli and Klebsiella were the most common causative agents. The mortality from medical management alone was 50%, medical management combined with emergency nephrectomy was 25% and medical management combined with percutaneous drainage was 13.5%. Mortality was significantly less in patients undergoing percutaneous drainage compared to other treatments (Pearson chi-square p0.001). Of the patients who underwent medical treatment with percutaneous drainage a small number (15) underwent elective nephrectomy and mortality was 6.6% (1 of 15).Percutaneous drainage should be part of the initial management strategy for emphysematous pyelonephritis. This strategy is associated with a lower mortality than medical management or emergency nephrectomy. Delayed elective nephrectomy may be required in some patients.

Background Aphids are phloem-feeding insects that cause significant economic losses to agriculture worldwide. While feeding and probing these insects deliver molecules, called effectors, inside their host to enable infestation. The identification and characterization of these effectors from different species that vary in their host range is an important step in understanding the infestation success of aphids and aphid host range variation. This study employs a multi-disciplinary approach based on transcriptome sequencing and proteomics to identify and compare effector candidates from the broad host range aphid Myzus persicae (green peach aphid) (genotypes O, J and F), and narrow host range aphids Myzus cerasi (black cherry aphid) and Rhopalosiphum padi (bird-cherry oat aphid). Results Using a combination of aphid transcriptome sequencing on libraries derived from head versus body tissues as well as saliva proteomics we were able to predict candidate effectors repertoires from the different aphid species and genotypes. Among the identified conserved or core effector sets, we identified a significant number of previously identified aphid candidate effectors indicating these proteins may be involved in general infestation strategies. Moreover, we identified aphid candidate effector sequences that were specific to one species, which are interesting candidates for further validation and characterization with regards to species-specific functions during infestation. We assessed our candidate effector repertoires for evidence of positive selection, and identified 49 candidates with DN/DS ratios >1. We noted higher rates of DN/DS ratios in predicted aphid effectors than non-effectors. Whether this reflects positive selection due to co-evolution with host plants, or increased neofunctionalization upon gene duplication remains to be investigated. Conclusion Our work provides a comprehensive overview of the candidate effector repertoires from three different aphid species with varying host ranges. Comparative analyses revealed candidate effectors that are most likely are involved in general aspects of infestation, whereas others, that are highly divergent, may be involved in specific processes important for certain aphid species. Insights into the overlap and differences in aphid effector repertoires are important in understanding how different species successfully infest different ranges of plant species. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2496-6) contains supplementary material, which is available to authorized users.

This paper reports findings from a follow-up evaluation of a short inservice course designed to enable teachers to assess and teach pupils with PMLD more effectively by combining the use of custom built ICT hardware and software (the Declan materials) and cognitive developmental psychology. The Declan materials were introduced to teachers at a short inservice course and all participants completed an evaluation form at the end of the course. Twelve months later participants took part in a telephone interview. Just under half of those interviewed were using the materials with pupils and were very positive about the impact of the materials on pupil progress. Those not using the materials were still positive about the course, but reported a number of barriers to use of the materials, including lack of confidence in setting up the hardware and software, lack of other resources, and classroom organizational issues.

Purpose: The aim of this study was to critically appraise the efficacy and complications of Therapeutic Transarterial Embolisation (TAE) in various benign and malignant renal conditions. Material and Methods: The records of all patients who underwent renal embolisation procedures, at a single institution, between March 1992 and March 2004, were reviewed. The patients were identified from hospital records via the procedure coding system and the radiology department procedures book and were analysed retrospectively. Twenty-nine patients were analysed, looking at indications, clinical outcome, complications and long-term results. Results: Twenty-nine patients underwent 35 embolisation procedures during this period. Fourteen patients with benign diseases underwent 17 embolisation procedures for haematuria or intractable pain. In the haematuria group, selective embolisation was used to treat bleeding post percutaneous nephrolithotomy (PCNL) (n=4), angiomyolipoma (n=2), arteriovenous (AV) malformation (n=1), renal artery aneurysm (n=1) and renal trauma (n=2). In the renal pain group (n=3), non-selective embolisation was done. Two of these patients had recurrence of pain despite repeat embolisation and subsequently underwent nephrectomy. Fifteen patients with advanced renal malignancy, who were deemed unfit for surgery, underwent 18 embolisation procedures for symptomatic haematuria. Twelve of the 15 patients had successful outcomes with cessation of haematuria. Three patients required repeat embolisation procedures for continuing haematuria with success. There were no major embolisation-related complications. Minor complications were self-limiting and settled with conservative management. Conclusion: Renal artery embolisation is effective in managing haematuria in benign and malignant renal conditions where indicated, with minor and easily treatable adverse effects

Some plant-parasitic nematodes need to spend a significant part of their life cycles within plants while others have long-lasting biotrophic interactions with their hosts. In each case, these nematodes will need to suppress host defences. Large numbers of candidate effectors – proteins that nematodes introduce into their hosts – have been identified from a variety of nematode species as a result of genome and transcriptome projects. There is now a focus on functional characterization of these proteins. This has led to the identification of nematode proteins that suppress different types of host defence responses. Here we review progress towards understanding how nematodes manipulate host defences and how hormone signalling related to defence responses is targeted by nematodes.

Cotton, James A. et al., [Background] Globodera pallida is a devastating pathogen of potato crops, making it one of the most economically important plant parasitic nematodes. It is also an important model for the biology of cyst nematodes. Cyst nematodes and root-knot nematodes are the two most important plant parasitic nematode groups and together represent a global threat to food security., [Results] We present the complete genome sequence of G. pallida, together with transcriptomic data from most of the nematode life cycle, particularly focusing on the life cycle stages involved in root invasion and establishment of the biotrophic feeding site. Despite the relatively close phylogenetic relationship with root-knot nematodes, we describe a very different gene family content between the two groups and in particular extensive differences in the repertoire of effectors, including an enormous expansion of the SPRY domain protein family in G. pallida, which includes the SPRYSEC family of effectors. This highlights the distinct biology of cyst nematodes compared to the root-knot nematodes that were, until now, the only sedentary plant parasitic nematodes for which genome information was available. We also present in-depth descriptions of the repertoires of other genes likely to be important in understanding the unique biology of cyst nematodes and of potential drug targets and other targets for their control., [Conclusions] The data and analyses we present will be central in exploiting post-genomic approaches in the development of much-needed novel strategies for the control of G. pallida and related pathogens., We thank the many people at the Wellcome Trust Sanger Insitute who contributed to this work: Mark Simmonds, Natalie Smerdon, Dave Willey, Michael Quail, Matthew Jones and Richard Rance provided technical support in generating sequencing libraries; Robert Davies and Quan Lin arranged for data release to the SRA; Martin Aslett and Jacqueline Keane provided informatics support; Christine Lloyd assisted with sequencing management; Karen Brooks, Richard Clark, Olivia Lambert, Sarah Nichol and Alan Tracey assisted with curation and annotation of gene models. We thank Jennie Hibbard and Beverley Merry at the University of Leeds who provided technical support in generating biological material.

This project was funded through BBSRC grants BB/F00334X/1 and BB/H000801/1, Scottish Government Contract Research Fund project CRF0926 and through a BBSRC CASE award for which the industrial partner was Mylnefield Research Services. The James Hutton Institute receives funding from the Scottish Government. EG was funded through ERASMUS MUNDUS programme 2008–102 (EUMAINE). Background: The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. Results: The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. Conclusion: This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process. Publisher PDF

Background Endovascular stents have been suggested as a means to improve the patency of arteries after angioplasty in patients with intermittent claudication. Objectives The null hypothesis to be tested by this review is that for individuals with claudication, the use of an endovascular stent in addition to percutaneous transluminal angioplasty, does not improve symptoms of life-style limiting claudication, when compared to percutaneous angioplasty alone. Search strategy We searched the Specialised Register of the Cochrane Peripheral Vascular Disease Group, (last searched October 2002), and the Cochrane Central Register of Controlled Trials (CENTRAL), (last searched Issue 3, 2002). We also searched MEDLINE and EMBASE (up to and including January 2001); bibliographies of published data, and hand searched the Journal of Vascular Interventional Radiology (1990-2001). Enquiries were made to stent manufacturers for unpublished trial data. Selection criteria Randomised trials comparing angioplasty alone, versus angioplasty with endovascular stents, in subjects with intermittent claudication. Data collection and analysis Both reviewers independently assessed trial quality and extracted the data. Only published trial data were used. Effectiveness was measured by pre-defined primary outcome measures: restenosis / reocclusion rates and maximum walking distance. Main results Two trials were included with a total sample size of 104 subjects. Both trials included only individuals with femoro-popliteal disease and compared angioplasty and stenting with the Palmaz stent against angioplasty alone. Although one study showed a slight statistical advantage in arterial patency after angioplasty alone, this was not found when the two studies were combined. No differences in the secondary outcomes in either study were detected. Reviewer's conclusions The small number of relevant trials identified, together with the small sample sizes and methodological weaknesses, severely limit the usefulness of this review in guiding practice. Larger multicentre trials are needed.

Introduction: The management of occlusive femoropopliteal disease continues to evolve and a definitive strategy remains to be defined. We examine the utility of subintimal angioplasty (SIA) in our institution. Methods: A retrospective study with predefined end-points, including technical success and primary patency. Results: 61 consecutive cases were identified (claudicants n=29 and critical ischaemia n=32). Sixty-four percent of occlusions were greater than 10cm with poor run-off (60% with two vessels or less). Technical and physiological success was 95% and 79% respectively, with clinical improvement reported by 72%. At a mean follow-up of 20 months twelve-month primary patency (assessed clinically, with ABPI and selective duplex scanning) was 67% (subgroup analysis: claudicants 83%, criticals 53%, p=0.02) and morbidity 8% with no limb loss or procedure related mortality. Conclusion: SIA is an effective procedure for chronic lower limb ischaemia with acceptable outcome. Our experience correlates well with evidence in the current literature.

Spontaneous perirenal haemorrhage is a rare but potentially life-threatening condition and requires urgent management. Diagnosis is based on clinical assessment combined with radiological investigations, as reported by Belville et al. (Radiology 172:733, 1989) and Zagoria et al. (J Urol 145:468, 1991). Reported is a case of spontaneous perirenal haemorrhage diagnosed on computed tomography scan and then successfully embolized using coils.