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Low-intensity pulsed ultrasound (LIPUS) treatment of fractures has been available to the orthopaedic community for nearly three decades; however, it is still considered an experimental treatment by some clinicians, even though there is a wealth of clinical data. Based on the evaluation of clinical trial data, we have established key criteria which can lead to LIPUS success and avoid failure. These are fracture gap size and stability, accurate transducer placement and minimum treatment number. However, from a clinician’s view, the correct attitude to treatment must be observed, and this has also been discussed. It is hoped, armed with this new evaluation of the clinical data, that clinicians can treat patients with LIPUS more effectively, resulting in fewer failures of treatment.

ImportanceData on the epidemiology of mild to moderately severe COVID-19 are needed to inform public health guidance.ObjectiveTo evaluate associations between 2 or 3 doses of mRNA COVID-19 vaccine and attenuation of symptoms and viral RNA load across SARS-CoV-2 viral lineages.Design, Setting, and ParticipantsA prospective cohort study of essential and frontline workers in Arizona, Florida, Minnesota, Oregon, Texas, and Utah with COVID-19 infection confirmed by reverse transcriptase–polymerase chain reaction testing and lineage classified by whole genome sequencing of specimens self-collected weekly and at COVID-19 illness symptom onset. This analysis was conducted among 1199 participants with SARS-CoV-2 from December 14, 2020, to April 19, 2022, with follow-up until May 9, 2022, reported.ExposuresSARS-CoV-2 lineage (origin strain, Delta variant, Omicron variant) and COVID-19 vaccination status.Main Outcomes and MeasuresClinical outcomes included presence of symptoms, specific symptoms (including fever or chills), illness duration, and medical care seeking. Virologic outcomes included viral load by quantitative reverse transcriptase–polymerase chain reaction testing along with viral viability.ResultsAmong 1199 participants with COVID-19 infection (714 [59.5%] women; median age, 41 years), 14.0% were infected with the origin strain, 24.0% with the Delta variant, and 62.0% with the Omicron variant. Participants vaccinated with the second vaccine dose 14 to 149 days before Delta infection were significantly less likely to be symptomatic compared with unvaccinated participants (21/27 [77.8%] vs 74/77 [96.1%]; OR, 0.13 [95% CI, 0-0.6]) and, when symptomatic, those vaccinated with the third dose 7 to 149 days before infection were significantly less likely to report fever or chills (5/13 [38.5%] vs 62/73 [84.9%]; OR, 0.07 [95% CI, 0.0-0.3]) and reported significantly fewer days of symptoms (10.2 vs 16.4; difference, −6.1 [95% CI, −11.8 to −0.4] days). Among those with Omicron infection, the risk of symptomatic infection did not differ significantly for the 2-dose vaccination status vs unvaccinated status and was significantly higher for the 3-dose recipients vs those who were unvaccinated (327/370 [88.4%] vs 85/107 [79.4%]; OR, 2.0 [95% CI, 1.1-3.5]). Among symptomatic Omicron infections, those vaccinated with the third dose 7 to 149 days before infection compared with those who were unvaccinated were significantly less likely to report fever or chills (160/311 [51.5%] vs 64/81 [79.0%]; OR, 0.25 [95% CI, 0.1-0.5]) or seek medical care (45/308 [14.6%] vs 20/81 [24.7%]; OR, 0.45 [95% CI, 0.2-0.9]). Participants with Delta and Omicron infections who received the second dose 14 to 149 days before infection had a significantly lower mean viral load compared with unvaccinated participants (3 vs 4.1 log10 copies/μL; difference, −1.0 [95% CI, −1.7 to −0.2] for Delta and 2.8 vs 3.5 log10 copies/μL, difference, −1.0 [95% CI, −1.7 to −0.3] for Omicron).Conclusions and RelevanceIn a cohort of US essential and frontline workers with SARS-CoV-2 infections, recent vaccination with 2 or 3 mRNA vaccine doses less than 150 days before infection with Delta or Omicron variants, compared with being unvaccinated, was associated with attenuated symptoms, duration of illness, medical care seeking, or viral load for some comparisons, although the precision and statistical significance of specific estimates varied.

Funding: This work was partly funded by the Perry foundation (JAP) (https://www.perryfoundation.co.uk/), The Rural and Environment Science and Analytical Services (RESAS) division of the Scottish Government (JAP & JTJ) (https://www.gov.scot/) and BBSRC award BB/V00249X/1 (JAP, TS & JTJ) (https://www.ukri.org/councils/bbsrc/). Host-specific plant pathogens must coordinate their life cycles with the availability of a host plant. Although this is frequently achieved through a response to specific chemical cues derived from the host plant, little is known about the molecular basis of the response to such cues and how these are used to trigger activation of the life cycle. In host-specific plant-parasitic cyst nematodes, unhatched juvenile nematodes lie dormant in the eggshell until chemical cues from a suitable host plant are detected and the hatching process is initiated. The molecular mechanisms by which hatch is linked to the presence of these chemical cues is unknown. We have identified a novel annexin-like protein that is localised to the eggshell of the potato cyst nematode Globodera rostochiensis. This annexin is unique in having a short peptide insertion that structural modelling predicts is present in one of the calcium-binding sites of this protein. Host-induced gene silencing of the annexin impacts the ability of the nematode to regulate and control permeability of the eggshell. We show that in the presence of the chemicals that induce hatching annexin lipid binding capabilities change, providing the first molecular link between a nematode eggshell protein and host-derived cues. This work demonstrates how a protein from a large family has been recruited to play a critical role in the perception of the presence of a host and provides a new potential route for control of cyst nematodes that impact global food production. Publisher PDF

Funding: KL was funded by a BBSRC EASTBIO DTP studentship provided through the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) grant number BB/T00875X/1 and by the Rural and Environment Science and Analytical Services Division of the Scottish Government at The James Hutton Institute and The University of St Andrews. Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA licence 125034/359149/3 and funded by BBSRC grants BB/R011311/1, BB/N021908/1, and BB/S006397/1. Plant parasitic nematodes need to overcome the barrier presented by the plant cell wall in order to invade their host. A variety of plant cell wall degrading enzymes are present in endoparasitic nematodes including enzymes that degrade cellulose (beta 1,4 endoglucanases) and various pectin components. We describe the cloning and functional analysis of genes encoding GH53 arabinogalactan endo-1,4-beta-galactosidases from three related plant parasitic nematodes Globodera rostochiensis, Globodera pallida and Rotylenchulus reniformis. Phylogenetic and structural analyses strongly indicate that these genes have been acquired by horizontal gene transfer from bacteria. We show that the genes are expressed at invasive stages of the parasites in the secretory gland cells. We also demonstrate that the enzymes from these species are biochemically active, showing the expected hydrolytic enzymatic activity when galactan was used as a substrate. This work further demonstrates the importance of cell wall degradation to the success of the parasitic process and the extensive role that horizontal gene transfer has played in the evolution of plant parasitism by nematodes. Publisher PDF

Taxonomy Phylum Nematoda; class Chromadorea; order Rhabditida; suborder Tylenchina; infraorder Tylenchomorpha; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; Genus Globodera. Biology Potato cyst nematodes (PCN) are biotrophic, sedentary endoparasitic nematodes. Invasive (second) stage juveniles (J2) hatch from eggs in response to the presence of host root exudates and subsequently locate and invade the host. The nematodes induce the formation of a large, multinucleate syncytium in host roots, formed by fusion of up to 300 root cell protoplasts. The nematodes rely on this single syncytium for the nutrients required to develop through a further three moults to the adult male or female stage. This extended period of biotrophy—between 4 and 6 weeks in total—is almost unparalleled in plant–pathogen interactions. Females remain at the root while adult males revert to the vermiform body plan of the J2 and leave the root to locate and fertilize the female nematodes. The female body forms a cyst that contains the next generation of eggs. Host range The host range of PCN is limited to plants of the Solanaceae family. While the most economically important hosts are potato (Solanum tuberosum), tomato (Solanum lycopersicum), and aubergine (Solanum melongena), over 170 species of Solanaceae are thought to be potential hosts for PCN (Sullivan et al., 2007). Disease symptoms Symptoms are similar to those associated with nutrient deficiency, such as stunted growth, yellowing of leaves and reduced yields. This absence of specific symptoms reduces awareness of the disease among growers. Disease control Resistance genes (where available in suitable cultivars), application of nematicides, crop rotation. Great effort is put into reducing the spread of PCN through quarantine measures and use of certified seed stocks. Useful websites Genomic information for PCN is accessible through WormBase ParaSite., PCN are biotrophic pathogens with a survival stage that allows them to persist in soil in the absence of a host facilitating their spread to all regions where potatoes are grown.

Summary Potato cyst nematodes (PCN) are estimated to cause over £50 million worth of crop losses in the UK each year. It has been shown that the infective juveniles are able to alter their surface composition to avoid damage from host defence mechanisms. However, relatively few proteins present on the cuticle surface of PCN juveniles have been identified. We have developed a method based upon biotinylation that allows selective labelling of proteins present on the cuticle surface of PCN. Isolated proteins can consequently be affinity purified and identified using mass spectrometry proteomics. Using this technique, we identify a variety of proteins present on the surface of PCN, including all previously described PCN surface proteins. Identification of known surface coat proteins using these methods demonstrates the viability of the process for isolation of novel surface coat proteins. Subsequent analysis confirmed that the genes encoding seven of the novel proteins were expressed in the hypodermis. This work provides a technique for study of surface proteins in a wide range of nematodes and expands our knowledge of the surface proteome of PCN.

This work was supported by the Rural & Environment Science & Analytical Services Division of the Scottish Government, the BBSRC, through the joint projects CRF/2009/SCRI/SOP 0929, BB/L008025/1 and BB/K018299/1. Additional funding was obtained through the James Hutton Institute SEEDCORN initiative, AHDB Potato, the Perry Foundation and The Felix Cobbold Trust. Amanpreet Kaur was supported by the Commonwealth Scholarship Commission through a Commonwealth split-site Ph.D. grant. Key message: The nematode resistance gene H2 was mapped to the distal end of chromosome 5 in tetraploid potato. The H2 resistance gene, introduced into cultivated potatoes from the wild diploid species Solanum multidissectum, confers a high level of resistance to the Pa1 pathotype of the potato cyst nematode Globodera pallida. A cross between tetraploid H2-containing breeding clone P55/7 and susceptible potato variety Picasso yielded an F1 population that segregated approximately 1:1 for the resistance phenotype, which is consistent with a single dominant gene in a simplex configuration. Using genome reduction methodologies RenSeq and GenSeq, the segregating F1 population enabled the genetic characterisation of the resistance through a bulked segregant analysis. A diagnostic RenSeq analysis of the parents confirmed that the resistance in P55/7 cannot be explained by previously characterised resistance genes. Only the variety Picasso contained functionally characterised disease resistance genes Rpi-R1, Rpi-R3a, Rpi-R3b variant, Gpa2 and Rx, which was independently confirmed through effector vacuum infiltration assays. RenSeq and GenSeq independently identified sequence polymorphisms linked to the H2 resistance on the top end of potato chromosome 5. Allele-specific KASP markers further defined the locus containing the H2 gene to a 4.7 Mb interval on the distal short arm of potato chromosome 5 and to positions that correspond to 1.4 MB and 6.1 MB in the potato reference genome. Publisher PDF

Authors acknowledge funding from the Agriculture and Horticulture Development Board (KV) and The James Hutton Institute receives funding from the Rural and Environment Science and Analytical Services (RESAS) Division of the Scottish Government. Postprint

Summary This paper presents studies on the life cycle of Heterodera sacchari under in vitro conditions. Pluronic gel was used as a medium for growth of H. sacchari. The life cycle was completed in 7-9 weeks on rice (Oryza sativa, ‘Nipponbare’). After infection, juveniles developed and reached the reproducing adult female stage at 25 days post inoculation (dpi). At 35 dpi, all females produced eggs in various numbers. Some females were translucent and eggs inside could be counted. At 49 dpi females started to tan and developed into dark brown cysts. Hatching of H. sacchari juveniles from cysts could be stimulated by 3 mM ZnCl2 but not by rice root exudates. The in vitro culture of H. sacchari on Pluronic gel can be used efficiently to collect post-infective nematode/host samples at different time points for various studies and to screen different rice cultivars for resistance/susceptibility.

Take-up of employment programs among people with disabilities can be limited by the administrative burdens of decision-making, which must factor in the complexities of how work affects disability cash assistance payments. This study presents evidence on using outreach motivated by behavioral research to encourage enrollment in a pilot initiative with the Social Security Administration that simplified Social Security Disability Insurance payment rules. Because enrolling would leave some beneficiaries worse off, informed enrollment decisions required understanding both the complexities of current rules and potential effects of the new demonstration rules. We sought to counteract bottlenecks stemming from decision-making burdens through increased outreach with tailored messaging. A randomized controlled trial was used to test two features of a reminder postcard. First, we compared fold-over postcards containing information about the demonstration to open postcards with more generic information, finding that fold-over postcards increased enrollment by around 25 percent (or 0.12 percentage points). Second, we compared an urgent message framing with no stated enrollment end-date to a deadline framing with an explicit enrollment cutoff date. Although the final enrollment rate was similar across timeline framing options, the urgent framing appears to have resulted in faster enrollment.

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.

Plant parasitic nematodes are damaging pests on all crops grown across the world. They exploit plants using a range of strategies, ranging from simple browsing ectoparasitism to highly complex biotrophic endoparasites. Some nematodes induce the formation of complex feeding structures in the roots of their hosts that require extensive reprogramming of host gene expression. These changes include changes in fundamentally important plant processes, including the cell cycle. Natural resistance can be used to control plant nematodes, and great progress has been made in mapping and identifying resistance genes against nematodes. Recent work has shown that the dependence of nematodes on a feeding structure has allowed plants to evolve new mechanisms of resistance that target this structure with a toxic response.

Funding: Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA licence 125034/359149/3, and was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/R011311/1, BB/N021908/1, and BB/S006397/1, a Synthego Genome Engineer grant, and a Genewiz grant. A.A. was supported by a Wellcome/Newton trust Institutional Strategic Support Fund grant and a UKRI Future Leaders Fellowship MR/S033769/1. C.J.L. was supported by BBSRC grant BB/N016866/1. J.J. receives funding from the Scottish Government Rural and Environmental Science and Analytical Services division. T.R.M. and T.J.B. were supported by grants from the Iowa Soybean Association and by State of Iowa and Hatch funds. A.P.S. and S.A. were supported by grants RPG-2016-089 and BB/L019884/1. Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security. Publisher PDF

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

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and The James Hutton Institute through a PhD studentship to SE-vdA. The James Hutton Institute receives funding from the Scottish Government Rural and Environment Science and Analytical Services division. SE-vdA is supported by BBSRC grant BB/M014207/1. Sedentary Plant-Parasitic Nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active “feeding sites”. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-terminally Encoded Peptide (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant-parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests CEPs likely evolved de novo in R. reniformis. We have characterised the first member of this large gene family (RrCEP1), demonstrating its significant upregulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of upregulating plant nitrate transporter (AtNRT2.1) expression, while simultaneously reducing primary root elongation. When a non-CEP containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment a smaller feeding site is produced. We hypothesise that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species; 1) increasing host nitrate uptake while 2) limiting the size of the syncytial feeding site produced. Publisher PDF

This work was supported by the EU 7th Framework REPHRAME project (KBBE.2010.1.4-09) and by FEDER Funds through the Operational Programme for Competitiveness Factors – COMPETE and National Funds through FCT – Foundation for Science and Technology under the Strategic Projects PEst-C/AGR/UI0115/2011 and PEst-OE/AGR/UI0115/2014. ME is funded by the FCT (Fundação para a Ciência e a Tecnologia, IP) under the PhD grant (SFRH/BD/84541/2012). The James Hutton Institute receives funding from the Scottish Government. We have previously identified two secreted glutathione S-transferases (GST) expressed in the pharyngeal gland cell of Bursaphelenchus xylophilus, which are upregulated post infection of the host. This study examines the functional role of GSTs in B. xylophilus biology. We analysed the expression profiles of all predicted GSTs in the genome and the results showed that they belong to kappa and cytosolic subfamilies and the majority are upregulated post infection of the host. A small percentage is potentially secreted and none is downregulated post infection of the host. One secreted protein was confirmed as a functional GST and is within a cluster that showed the highest expression fold change in infection. This enzyme has a protective activity that may involve host defences, namely in the presence of terpenoid compounds and peroxide products. These results suggest that GSTs secreted into the host participate in the detoxification of host-derived defence compounds and enable successful parasitism. Postprint

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

This work was funded by National Funds through FCT – Foundation for Science and Technology under the Project UID/AGR/00115/2013. ME was funded by FCT under the PhD grant (SFRH/BD/84541/2012). The James Hutton Institute receives funding from the Rural and Environment Science and Analytical Services Division of the Scottish Government. This work was also supported by Hatch Act and State of Iowa funds. SE-vdA is supported by BBSRC grant BB/M014207/1 and BB/R011311/1. Background: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. Results: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel “pioneer” effectors. Conclusions: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda. Publisher PDF

This work was supported by the National Natural Science Foundation of China (Grant No. 31371922) and the Fundamental Research Funds for the Central Universities (KYZ201615). John T. Jones is supported by funding from the Rural and Environment Science and Analytical Services Division of the Scottish Government. Meloidogyne incognita is the most economically important plant-parasitic nematode. M. incognita manipulates plant cell development and metabolism by injecting effectors from the esophageal glands into the plant host. Chorismate mutase (CM) is one such effector that may contribute to successful parasitism by M. incognita. In this study, we identified and functionally characterized a novel CM effector, Mi-CM-3, which is more similar to CMs from bacteria than to those previously characterised from other phytoparasitic nematodes. Spatial and temporal expression assays showed that Mi-cm-3 mRNA accumulates specifically in the subventral esophageal glands, and that transcription is up-regulated during the early parasitic stages of the nematode. In planta gene silencing of Mi-cm-3 attenuated nematode parasitism. In addition, Mi-cm-3 could fully restore the full virulence phenotype of the pathogenic bacterium Xanthomonas oryzae pv. oryzae by complementation when it was introduced into a mutant strain carrying a deletion in the CM gene. Transient expression of Mi-CM-3 caused a reduction of SA and PR1 levels in Nicotiana benthamiana in response to oomycete pathogen Phytophthora capsici infection, while confocal observations showed that Mi-CM-3 was localized within the cytoplasm and the nucleus, but not the plastids, of transfected N. benthamiana leaf cells. Constitutive expression of Mi-CM-3 in N. benthamiana plants inhibited root growth and increased susceptibility to M. incognita infection. These results provide direct experimental evidence to show that Mi-CM-3 may play an important role in suppressing plant immunity by regulating the SA pathway during the early parasitic stage of M. incognita so as to promote nematode parasitism. Postprint

This work was supported by the REPHRAME project (KBBE.2010.1.4-09). ME is funded by FCT (Fundacao para a Ciencia e a Tecnologia, IP) under a PhD grant (SFRH/BD/84541/2012). ME and MM are also funded by FEDER Funds through the Operational Programme for Competitiveness Factors—COMPETE and National Funds through FCT—Foundation for Science and Technology under the Strategic Projects PEst-C/AGR/UI0115/2011 and PEst-OE/AGR/UI0115/2014. AS was funded by an ERASMUS MUNDUS Category B scholarship awarded through project 2008–2102 (EUMAINE). The James Hutton Institute receives funding from the Scottish Government Rural and Environmental Science and Analytical Services division (RESAS).

Summary Drastic physiological and morphological changes in parasites are crucial for the establishment of a successful infection. The nematode Bursaphelenchus xylophilus is the pathogenic agent of pine wilt disease, and little is known about the physiology and morphology in this nematode at the initial stage of infection. In this study, we devised an infection system using pine stem cuttings that allowed us to observe transcriptional and morphological changes in the host-infecting phytophagous phase. We found that 60 genes enriched in xenobiotic detoxification were up-regulated in two independent post-inoculation events, whereas down-regulation was observed in multiple members of collagen gene families. After 48 h of inoculation, the tails in some of the adult females exposed to the host changed in morphology. These results suggest that B. xylophilus may change its physiology and morphology to protect itself and to adapt to the host pine wood environment.

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.

Several structures associated with feeding by plant-parasitic nematodes have been described using two terms, feeding tubes and feeding plugs. However, both of these terms encompass multiple structures of independent evolution, some of which are functionally distinct. We have reviewed the literature on both structures and provide a new perspective on the function of intracellular feeding tubes to maintain the integrity and efficacy of the feeding site. We propose that they provide sufficient hydraulic resistance against the feeding site pressure to prevent it from collapsing during feeding. In addition, we propose that extracellular feeding tubes of migratory ectoparasites should be considered as the functional analogue of the stylet of all other plant-parasitic nematodes for withdrawal of host cell cytoplasm and, therefore, provide an example of convergent evolution. We also suggest that the main role of the feeding plug, irrespective of origin or composition, may be in adhesion.

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

Plant parasitism has evolved independently on at least four separate occasions in the phylum Nematoda. The application of next-generation sequencing (NGS) to plant-parasitic nematodes has allowed a wide range of genome- or transcriptome-level comparisons, and these have identified genome adaptations that enable parasitism of plants. Current genome data suggest that horizontal gene transfer, gene family expansions, evolution of new genes that mediate interactions with the host, and parasitism-specific gene regulation are important adaptations that allow nematodes to parasitize plants. Sequencing of a larger number of nematode genomes, including plant parasites that show different modes of parasitism or that have evolved in currently unsampled clades, and using free-living taxa as comparators would allow more detailed analysis and a better understanding of the organization of key genes within the genomes. This would facilitate a more complete understanding of the way in which parasitism has shaped the genomes of plant-parasitic nematodes.

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

The aim of this review was to undertake a survey of researchers working with plant-parasitic nematodes in order to determine a ‘top 10’ list of these pathogens based on scientific and economic importance. Any such list will not be definitive as economic importance will vary depending on the region of the world in which a researcher is based. However, care was taken to include researchers from as many parts of the world as possible when carrying out the survey. The top 10 list emerging from the survey is composed of: (1) root-knot nematodes (Meloidogyne spp.); (2) cyst nematodes (Heterodera and Globodera spp.); (3) root lesion nematodes (Pratylenchus spp.); (4) the burrowing nematode Radopholus similis; (5) Ditylenchus dipsaci; (6) the pine wilt nematode Bursaphelenchus xylophilus; (7) the reniform nematode Rotylenchulus reniformis; (8) Xiphinema index (the only virus vector nematode to make the list); (9) Nacobbus aberrans; and (10) Aphelenchoides besseyi. The biology of each nematode (or nematode group) is reviewed briefly.

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are major pests of potatoes. The G. pallida (and G. rostochiensis) life cycle includes both diapause and quiescent stages. Nematodes in dormancy (diapause or quiescent) are adapted for long-term survival and are more resistant to nematicides. This study analysed the mechanisms underlying diapause and quiescence. The effects of several compounds (8Br-cGMP, oxotremorine and atropine) on the activation of hatching were studied. The measurements of some morphometric parameters in diapaused and quiescent eggs after exposure to PRD revealed differences in dorsal gland length, subventral gland length and dorsal gland nucleolus. In addition, the expression of 2 effectors (IVg9 and cellulase) was not induced in diapaused eggs in water or PRD, while expression was slightly induced in quiescent eggs. Finally, we performed a comparative study to identify orthologues of C. elegans diapause related genes in plant-parasitic nematodes (G. pallida, Meloidogyne incognita, M. hapla and Bursaphelenchus xylophilus). This analysis suggested that it was not possible to identify G. pallida orthologues of the majority of C. elegans genes involved in the control of dauer formation. All these data suggest that G. pallida may use different mechanisms to C. elegans in regulating the survival stage., The authors thank the Spanish Ministry of Education for funding to the fi rst author under the ‘ Ayudas para la movilidadpostdoctoralen centrosextranjeros ’ scheme. The James Hutton Institute receives funding from the Scottish Government. Statistical advice from Jim McNicol is gratefully acknowledged (BIOSS, UK).

Potato is produced on all continents except Antarctica and is the world’s third most important food crop. Potato production has increased dramatically in developing countries in the past two decades, and has now overtaken that in the developed world, underlining the growing importance of potato as a staple food crop to meet the demands of increasing human populations. Potato is also an important source of starch. It has been adapted for cultivation in a wide range of environments and, with the availability of significant germplasm resources, the potential to further exploit its natural biodiversity is considerable. Potato yields vary considerably across the world, with the lowest being in Sub-Saharan Africa

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs—"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist”—that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities., The education outreach programs “Present Your PhD Thesis to a 12-Year-Old” and “Shadow a Scientist” provide opportunities for scientists to improve science communication skills and for students to learn about research.