Publications by authors named "Nicholas Talbot"

154 Publications

Selective MAP1LC3C (LC3C) autophagy requires noncanonical regulators and the C-terminal peptide.

J Cell Biol 2021 Jul 14;220(7). Epub 2021 May 14.

Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH.

LC3s are canonical proteins necessary for the formation of autophagosomes. We have previously established that two paralogs, LC3B and LC3C, have opposite activities in renal cancer, with LC3B playing an oncogenic role and LC3C a tumor-suppressing role. LC3C is an evolutionary late gene present only in higher primates and humans. Its most distinct feature is a C-terminal 20-amino acid peptide cleaved in the process of glycine 126 lipidation. Here, we investigated mechanisms of LC3C-selective autophagy. LC3C autophagy requires noncanonical upstream regulatory complexes that include ULK3, UVRAG, RUBCN, PIK3C2A, and a member of ESCRT, TSG101. We established that postdivision midbody rings (PDMBs) implicated in cancer stem-cell regulation are direct targets of LC3C autophagy. LC3C C-terminal peptide is necessary and sufficient to mediate LC3C-dependent selective degradation of PDMBs. This work establishes a new noncanonical human-specific selective autophagic program relevant to cancer stem cells.
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http://dx.doi.org/10.1083/jcb.202004182DOI Listing
July 2021

Investigating the cell and developmental biology of plant infection by the rice blast fungus Magnaporthe oryzae.

Fungal Genet Biol 2021 Apr 18:103562. Epub 2021 Apr 18.

The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich NR47UH, United Kingdom. Electronic address:

Magnaporthe oryzae is the causal agent of rice blast disease, the most widespread and serious disease of cultivated rice. Live cell imaging and quantitative 4D image analysis have provided new insight into the mechanisms by which the fungus infects host cells and spreads rapidly in plant tissue. In this video review article, we apply live cell imaging approaches to understanding the cell and developmental biology of rice blast disease. To gain entry to host plants, M. oryzae develops a specialised infection structure called an appressorium, a unicellular dome-shaped cell which generates enormous turgor, translated into mechanical force to rupture the leaf cuticle. Appressorium development is induced by perception of the hydrophobic leaf surface and nutrient deprivation. Cargo-independent autophagy in the three-celled conidium, controlled by cell cycle regulation, is essential for appressorium morphogenesis. Appressorium maturation involves turgor generation and melanin pigment deposition in the appressorial cell wall. Once a threshold of turgor has been reached, this triggers re-polarisation which requires regulated generation of reactive oxygen species, to facilitate septin GTPase-dependent cytoskeletal re-organisation and re-polarisation of the appressorium to form a narrow, rigid penetration peg. Infection of host tissue requires a further morphogenetic transition to a pseudohyphal-type of growth within colonised rice cells. At the same time the fungus secretes an arsenal of effector proteins to suppress plant immunity. Many effectors are secreted into host cells directly, which involves a specific secretory pathway and a specialised structure called the biotrophic interfacial complex. Cell-to-cell spread of the fungus then requires development of a specialised structure, the transpressorium, that is used to traverse pit field sites, allowing the fungus to maintain host cell membrane integrity as new living plant cells are invaded. Thereafter, the fungus rapidly moves through plant tissue and host cells begin to die, as the fungus switches to necrotrophic growth and disease symptoms develop. These morphogenetic transitions are reviewed in the context of live cell imaging studies.
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http://dx.doi.org/10.1016/j.fgb.2021.103562DOI Listing
April 2021

Chitosan inhibits septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae in a protein kinase C and Nox1 NADPH oxidase-dependent manner.

New Phytol 2021 05 17;230(4):1578-1593. Epub 2021 Mar 17.

School of Biosciences, University of Exeter, Exeter,, EX4 4QD, UK.

Chitosan is a partially deacetylated linear polysaccharide composed of β-1,4-linked units of d-glucosamine and N-acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway, the Mps1 mitogen-activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin-mediated plant infection.
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http://dx.doi.org/10.1111/nph.17268DOI Listing
May 2021

Protein glycosylation during infection by plant pathogenic fungi.

New Phytol 2021 05 10;230(4):1329-1335. Epub 2021 Feb 10.

State Key Laboratory of Agricultural Microbiology and Provincial Hubei Key Laboratory of Plant Pathology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.

Glycosylation is a conserved set of post-translational modifications that exists in all eukaryotic cells. During the last decade, the role of glycosylation in plant pathogenic fungi has received significant attention and considerable progress has been made especially in Ustilago maydis and Magnaporthe oryzae. Here, we review recent advances in our understanding of the role of N-glycosylation, O-glycosylation and glycosylphosphatidylinositol (GPI) anchors during plant infection by pathogenic fungi. We highlight the roles of these processes in regulatory mechanisms associated with appressorium formation, host penetration, biotrophic growth and immune evasion. We argue that improved knowledge of glycosylation pathways and the impact of these modifications on fungal pathogenesis is overdue and could provide novel strategies for disease control.
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http://dx.doi.org/10.1111/nph.17207DOI Listing
May 2021

The contribution of commercial fireworks to both local and personal air quality in Auckland, New Zealand.

Environ Sci Pollut Res Int 2021 May 7;28(17):21650-21660. Epub 2021 Jan 7.

School of Chemical Sciences, University of Auckland, Auckland, New Zealand.

Firework displays produce large amounts of particulate matter (PM), contributing to poor air quality in local areas. Since short-term exposure to particulate matter correlates with increased mortality risks, these celebrations may impact both human health and the environment. Little is known about the particulate matter produced from recreational fireworks, as most studies have focused on professional large-scale events. In New Zealand, it is common for consumer fireworks to be ignited within residential areas during the Guy Fawkes celebration around 5 November. To better understand the contribution of individual fireworks on local air quality, ambient PM sampling was conducted in the 10 days surrounding Guy Fawkes Day in Auckland, New Zealand. These data were supplemented with measurements of firework emissions from 11 different individual products, including smoke bombs, sparklers, and Roman candles. Filter sampling results indicated that personal fireworks can contribute to ground level ambient air quality during celebrations, increasing ambient PM concentrations by 21.6 μg m over a 12-h sampling period. The use of personal fireworks can expose consumers to PM concentrations much higher, up to 9.51 mg m from individual sparkler use under worst-case scenario assumptions. The inhalation of sparkler emissions for just 8 min can lead to an exposure to PM mass greater than that from daily recommended limits (50 μg m exposure over 24 h). X-ray fluorescence (XRF) analysis indicated that potassium (K) and strontium (Sr) can be used as tracers for local firework use and that arsenic (As) may be an important contaminant during Guy Fawkes celebrations. The PM from personal fireworks contained large amounts of chlorine (Cl), which may be indicative of perchlorate oxidizers. In addition, lead (Pb) was observed in the PM generated from two of the colored sparklers, which were marketed as "safer" alternatives to more explosive firework products.
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http://dx.doi.org/10.1007/s11356-020-11889-4DOI Listing
May 2021

Getting a grip on blast.

Nat Microbiol 2020 12;5(12):1457-1458

The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, UK.

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http://dx.doi.org/10.1038/s41564-020-00821-8DOI Listing
December 2020

Tobacco smoking induces metabolic reprogramming of renal cell carcinoma.

J Clin Invest 2021 Jan;131(1)

Department of Cancer Biology and.

BACKGROUNDClear cell renal cell carcinoma (ccRCC) is the most common histologically defined renal cancer. However, it is not a uniform disease and includes several genetic subtypes with different prognoses. ccRCC is also characterized by distinctive metabolic reprogramming. Tobacco smoking (TS) is an established risk factor for ccRCC, with unknown effects on tumor pathobiology.METHODSWe investigated the landscape of ccRCCs and paired normal kidney tissues using integrated transcriptomic, metabolomic, and metallomic approaches in a cohort of white males who were long-term current smokers (LTS) or were never smokers (NS).RESULTSAll 3 Omics domains consistently identified a distinct metabolic subtype of ccRCCs in LTS, characterized by activation of oxidative phosphorylation (OXPHOS) coupled with reprogramming of the malate-aspartate shuttle and metabolism of aspartate, glutamate, glutamine, and histidine. Cadmium, copper, and inorganic arsenic accumulated in LTS tumors, showing redistribution among intracellular pools, including relocation of copper into the cytochrome c oxidase complex. A gene expression signature based on the LTS metabolic subtype provided prognostic stratification of The Cancer Genome Atlas ccRCC tumors that was independent of genomic alterations.CONCLUSIONThe work identified the TS-related metabolic subtype of ccRCC with vulnerabilities that can be exploited for precision medicine approaches targeting metabolic pathways. The results provided rationale for the development of metabolic biomarkers with diagnostic and prognostic applications using evaluation of OXPHOS status. The metallomic analysis revealed the role of disrupted metal homeostasis in ccRCC, highlighting the importance of studying effects of metals from e-cigarettes and environmental exposures.FUNDINGDepartment of Defense, Veteran Administration, NIH, ACS, and University of Cincinnati Cancer Institute.
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http://dx.doi.org/10.1172/JCI140522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773408PMC
January 2021

Discovery of broad-spectrum fungicides that block septin-dependent infection processes of pathogenic fungi.

Nat Microbiol 2020 12 21;5(12):1565-1575. Epub 2020 Sep 21.

State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Key Laboratory of Major Crop Diseases & Collaborative Innovation Center for Hybrid Rice in Yangtze River Basin, Rice Research Institute, Sichuan Agricultural University, Chengdu, China.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.
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http://dx.doi.org/10.1038/s41564-020-00790-yDOI Listing
December 2020

Successful awake proning is associated with improved clinical outcomes in patients with COVID-19: single-centre high-dependency unit experience.

BMJ Open Respir Res 2020 09;7(1)

Oxford Centre for Respiratory Medicine, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.

The SARS-CoV-2 can lead to severe illness with COVID-19. Outcomes of patients requiring mechanical ventilation are poor. Awake proning in COVID-19 improves oxygenation, but on data clinical outcomes is limited. This single-centre retrospective study aimed to assess whether successful awake proning of patients with COVID-19, requiring respiratory support (continuous positive airways pressure (CPAP) or high-flow nasal oxygen (HFNO)) on a respiratory high-dependency unit (HDU), is associated with improved outcomes. HDU care included awake proning by respiratory physiotherapists. Of 565 patients admitted with COVID-19, 71 (12.6%) were managed on the respiratory HDU, with 48 of these (67.6%) requiring respiratory support. Patients managed with CPAP alone 22/48 (45.8%) were significantly less likely to die than patients who required transfer onto HFNO 26/48 (54.2%): CPAP mortality 36.4%; HFNO mortality 69.2%, (p=0.023); however, multivariate analysis demonstrated that increasing age and the inability to awake prone were the only independent predictors of COVID-19 mortality. The mortality of patients with COVID-19 requiring respiratory support is considerable. Data from our cohort managed on HDU show that CPAP and awake proning are possible in a selected population of COVID-19, and may be useful. Further prospective studies are required.
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http://dx.doi.org/10.1136/bmjresp-2020-000678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490910PMC
September 2020

Arsenic in Hair as a Marker of Exposure to Smoke from the Burning of Treated Wood in Domestic Wood Burners.

Int J Environ Res Public Health 2020 06 2;17(11). Epub 2020 Jun 2.

Institute of Geological and Nuclear Sciences, PO Box 30-368, Lower Hutt 5040, New Zealand.

Timber treated with the anti-fungal chemical copper chrome arsenate is used extensively in the New Zealand building industry. While illegal, the burning of treated timber is commonplace in New Zealand and presents a health risk. Outdoor ambient monitoring of arsenic in airborne particulate matter in New Zealand has identified levels that exceed the maximum standards of 5.5 ng m (annual average) at some urban locations. In this study, two-week-old beard hair samples were collected during the winter months to establish individual exposure to arsenic using Inductively Coupled Plasma-Mass Spectrometry. These results were then compared with questionnaire data about wood burner use for the two weeks prior to sampling, and spatial trends in arsenic from ambient monitoring. Results suggest that the burning of construction timber that may contain arsenic is associated with a higher level of arsenic in hair than those who burn logs or coal exclusively. There is no association between the area-level density of wood burners and arsenic levels but a significant correlation with individual household choice of fuel as well as the smell of wood smoke in the community, suggesting very localised influences. Strategies are needed to raise awareness of the risks of burning treated timber and to provide economically-viable alternatives.
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http://dx.doi.org/10.3390/ijerph17113944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312981PMC
June 2020

The sorting nexin FgAtg20 is involved in the Cvt pathway, non-selective macroautophagy, pexophagy and pathogenesis in Fusarium graminearum.

Cell Microbiol 2020 08 30;22(8):e13208. Epub 2020 Apr 30.

State Key Laboratory of Rice Biology and Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, China.

The sorting nexin Atg20/Snx42 plays an important role in autophagy. The wheat head blight pathogen Fusarium graminearum contains an FgAtg20 protein orthologous to Saccharomyces cerevisiae Atg20/Snx42, but its function remains largely unknown. Here, we report a role for FgAtg20 in regulating morphogenesis and fungal pathogenicity. Cytological observation and Western blot analysis revealed that ΔFgAtg20 mutants are defective in vacuolar transport and proteolysis of GFP-FgAtg8, indicating that FgAtg20 is required for non-selective macroautophagy. Furthermore, we found that FgATG20 is necessary for the maturation of FgApe1, an indicator of the cytoplasm-to-vacuole targeting (Cvt) pathway. Immunoblot analysis displayed lower level of FgPex14, a peroxisomal integral membrane protein in ΔFgAtg20 mutants, suggesting that pexophagy is impaired. Furthermore, we demonstrate that FgAtg20 forms a complex with FgAtg1, FgAtg11, FgAtg17 and FgAtg24. When considered together, we conclude that FgAtg20 plays a critical role in vegetative growth, conidiation and pathogenicity of the head blight pathogen, and is involved in the Cvt pathway, non-selective macroautophagy and pexophagy.
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http://dx.doi.org/10.1111/cmi.13208DOI Listing
August 2020

Publisher Correction: The glycogen synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal development and pathogenicity in Magnaporthe oryzae.

Sci Rep 2020 Jan 21;10(1):1187. Epub 2020 Jan 21.

Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-58148-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971253PMC
January 2020

Cycling in synchrony.

Elife 2019 12 2;8. Epub 2019 Dec 2.

The Sainsbury Laboratory, University of East Anglia, Norwich, United Kingdom.

The corn smut fungus uses two different mechanisms to control its cell cycle when it is infecting plants.
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http://dx.doi.org/10.7554/eLife.52884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887483PMC
December 2019

Optical coherence tomography (OCT) in unconscious and systemically unwell patients using a mobile OCT device: a pilot study.

BMJ Open 2019 11 7;9(11):e030882. Epub 2019 Nov 7.

Ophthalmology Department, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

Objective: This study aims to evaluate the feasibility of retinal imaging in critical care using a novel mobile optical coherence tomography (OCT) device. The Heidelberg SPECTRALIS FLEX module (Heidelberg Engineering, Heidelberg, Germany) is an OCT unit with a boom arm, enabling ocular OCT assessment in less mobile patients.

Design: We undertook an evaluation of the feasibility of using the SPECTRALIS FLEX for undertaking ocular OCT images in unconscious and critically ill patients.

Setting: This study was conducted in the critical care unit of a large tertiary referral unit in the United Kingdom.

Participants: 13 systemically unwell patients admitted to the critical care unit were purposively sampled to enable evaluation in patients with a range of clinical states.

Outcome Measures: The primary outcome was the feasibility of acquiring clinically interpretable OCT scans on a consecutive series of patients. The standardised scanning protocol included macula-focused OCT, OCT optic nerve head (ONH), OCT angiography (OCTA) of the macula and ONH OCTA.

Results: OCT images from 13 patients were attempted. The success rates of each scan type are 84% for OCT macula, 76% for OCT ONH, 56% for OCTA macula and 36% for OCTA ONH. The overall mean success rate of scans per patient was 64% (95% CI 46% to 81%). Clinicians reported clinical value in 100% scans which were successfully obtained, including both ruling in and ruling out relevant ocular complications such as corneal thinning, macular oedema and optic disc swelling. The most common causes of failure to achieve clinically interpretable scans were inadequately sustained OCT alignment in delirious patients and a compromised ocular surface due to corneal exposure.

Conclusions: This prospective evaluation indicates the feasibility and potential clinical value of the SPECTRALIS FLEX OCT system on the critical care unit. Portable OCT systems have the potential to bring instrument-based ophthalmic assessment to critically ill patients, enabling detection and micron-level monitoring of ocular complications.
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http://dx.doi.org/10.1136/bmjopen-2019-030882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858135PMC
November 2019

A sensor kinase controls turgor-driven plant infection by the rice blast fungus.

Nature 2019 10 9;574(7778):423-427. Epub 2019 Oct 9.

Department of Biosciences, University of Exeter, Exeter, UK.

The blast fungus Magnaporthe oryzae gains entry to its host plant by means of a specialized pressure-generating infection cell called an appressorium, which physically ruptures the leaf cuticle. Turgor is applied as an enormous invasive force by septin-mediated reorganization of the cytoskeleton and actin-dependent protrusion of a rigid penetration hypha. However, the molecular mechanisms that regulate the generation of turgor pressure during appressorium-mediated infection of plants remain poorly understood. Here we show that a turgor-sensing histidine-aspartate kinase, Sln1, enables the appressorium to sense when a critical turgor threshold has been reached and thereby facilitates host penetration. We found that the Sln1 sensor localizes to the appressorium pore in a pressure-dependent manner, which is consistent with the predictions of a mathematical model for plant infection. A Δsln1 mutant generates excess intracellular appressorium turgor, produces hyper-melanized non-functional appressoria and does not organize the septins and polarity determinants that are required for leaf infection. Sln1 acts in parallel with the protein kinase C cell-integrity pathway as a regulator of cAMP-dependent signalling by protein kinase A. Pkc1 phosphorylates the NADPH oxidase regulator NoxR and, collectively, these signalling pathways modulate appressorium turgor and trigger the generation of invasive force to cause blast disease.
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http://dx.doi.org/10.1038/s41586-019-1637-xDOI Listing
October 2019

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BMC Genomics 2019 Jul 23;20(1):605. Epub 2019 Jul 23.

Department of Systematic and Evolutionary Botany, University of Zurich, Zurich, Switzerland.

Background: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.

Results: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting.

Conclusions: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.
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http://dx.doi.org/10.1186/s12864-019-5629-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652019PMC
July 2019

The ORFeome: A Functional Genomics Community Resource.

Mol Plant Microbe Interact 2019 Dec 14;32(12):1564-1570. Epub 2019 Oct 14.

Biosciences, University of Exeter, Exeter EX4 4QD, U.K.

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for , which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors ( = 410) and secondary metabolite biosynthetic proteins ( = 171) in addition to genes residing at dispensable chromosomes ( = 122) or those that are preferentially expressed during plant infection ( = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of .[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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http://dx.doi.org/10.1094/MPMI-05-19-0123-ADOI Listing
December 2019

Leucine biosynthesis is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Curr Genet 2020 Feb 1;66(1):155-171. Epub 2019 Jul 1.

State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, People's Republic of China.

The rice blast fungus Magnaporthe oryzae causes one of the most devastating crop diseases world-wide and new control strategies for blast disease are urgently required. We have used insertional mutagenesis in M. oryzae to define biological processes that are critical for blast disease. Here, we report the identification of LEU2A by T-DNA mutagenesis, which putatively encodes 3-isopropylmalate dehydrogenase (3-IPMDH) required for leucine biosynthesis, implicating that synthesis of this amino acid is required for fungal pathogenesis. M. oryzae contains a further predicted 3-IPMDH gene (LEU2B), two 2-isopropylmalate synthase (2-IPMS) genes (LEU4 and LEU9) and an isopropylmalate isomerase (IPMI) gene (LEU1). Targeted gene deletion mutants of LEU1, LEU2A or LEU4 are leucine auxotrophs, and severely defective in pathogenicity. All phenotypes associated with mutants lacking LEU1, LEU2A or LEU4 could be overcome by adding exogenous leucine. The expression levels of LEU1, LEU2A or LEU4 genes were significantly down-regulated by deletion of the transcription factor gene LEU3, an ortholog of Saccharomyces cerevisiae LEU3. We also functionally characterized leucine biosynthesis genes in the wheat pathogen Fusarium graminearum and found that FgLEU1, FgLEU3 and FgLEU4 are essential for wheat head blight disease, suggesting that leucine biosynthesis in filamentous fungal pathogens may be a conserved factor for fungal pathogenicity and, therefore, a potential target for disease control.
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http://dx.doi.org/10.1007/s00294-019-01009-2DOI Listing
February 2020

Plant health emergencies demand open science: Tackling a cereal killer on the run.

PLoS Biol 2019 06 3;17(6):e3000302. Epub 2019 Jun 3.

Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh.

Outbreaks of emerging plant diseases and insect pests are increasing at an alarming rate threatening the food security needs of a booming world population. The role of plant pathologists in addressing these threats to plant health is critical. Here, we share our personal experience with the appearance in Bangladesh of a destructive new fungal disease called wheat blast and stress the importance of open-science platforms and crowdsourced community responses in tackling emerging plant diseases. Benefits of the open-science approach include recruitment of multidisciplinary experts, application of cutting-edge methods, and timely replication of data analyses to increase the robustness of the findings. Based on our experiences, we provide some general recommendations and practical guidance for responding to emerging plant diseases.
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http://dx.doi.org/10.1371/journal.pbio.3000302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6564034PMC
June 2019

Environment-dependent fitness gains can be driven by horizontal gene transfer of transporter-encoding genes.

Proc Natl Acad Sci U S A 2019 03 6;116(12):5613-5622. Epub 2019 Mar 6.

Biosciences, Living Systems Institute, University of Exeter, EX4 4QD Exeter, United Kingdom;

Many microbes acquire metabolites in a "feeding" process where complex polymers are broken down in the environment to their subunits. The subsequent uptake of soluble metabolites by a cell, sometimes called osmotrophy, is facilitated by transporter proteins. As such, the diversification of osmotrophic microorganisms is closely tied to the diversification of transporter functions. Horizontal gene transfer (HGT) has been suggested to produce genetic variation that can lead to adaptation, allowing lineages to acquire traits and expand niche ranges. Transporter genes often encode single-gene phenotypes and tend to have low protein-protein interaction complexity and, as such, are potential candidates for HGT. Here we test the idea that HGT has underpinned the expansion of metabolic potential and substrate utilization via transfer of transporter-encoding genes. Using phylogenomics, we identify seven cases of transporter-gene HGT between fungal phyla, and investigate compatibility, localization, function, and fitness consequences when these genes are expressed in Using this approach, we demonstrate that the transporters identified can alter how fungi utilize a range of metabolites, including peptides, polyols, and sugars. We then show, for one model gene, that transporter gene acquisition by HGT can significantly alter the fitness landscape of We therefore provide evidence that transporter HGT occurs between fungi, alters how fungi can acquire metabolites, and can drive gain in fitness. We propose a "transporter-gene acquisition ratchet," where transporter repertoires are continually augmented by duplication, HGT, and differential loss, collectively acting to overwrite, fine-tune, and diversify the complement of transporters present in a genome.
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http://dx.doi.org/10.1073/pnas.1815994116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431176PMC
March 2019

Appressoria.

Curr Biol 2019 03;29(5):R144-R146

The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, NR4 7UH, UK. Electronic address:

Appressoria are specialised infection structures used by many disease-causing microorganisms to breach the outer surface of a host. Nick Talbot tells us about these small but incredibly powerful structures.
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http://dx.doi.org/10.1016/j.cub.2018.12.050DOI Listing
March 2019

Early outcomes and radiographic alignment of the Infinity total ankle replacement with a minimum of two year follow-up data.

Foot Ankle Surg 2019 Dec 22;25(6):826-833. Epub 2018 Nov 22.

The Royal Devon and Exeter NHS Foundation Trust, Barrack Road, Exeter, EX2 5DW, United Kingdom.

Background: The Infinity total ankle replacement (Wright Medical Technology, Memphis, TN) is a low profile, fluoroscopically navigated, fixed-bearing device. We hypothesised that the fluoroscopic navigation would allow more accurate alignment of the prosthesis than conventional techniques. We present our minimum two year follow up data of Infinity ankle replacements.

Methods: All total ankle replacements (TARs) performed at our institution were prospectively followed-up with EQ5-D and MOx-FQ scores as well as intra-operative radiation exposure and radiographic alignment data. Post-operative radiographs were used to measure the alignment of the prostheses. We identified 20 implants with minimum of two year follow up which were compared to a control group of 20 Zenith TAR's (Corin, Cirencester, UK).

Results: Intra-operative fluoroscopic navigation has allowed excellent alignment of all prostheses. Median deviations from 90° alignment to the anatomical axis of the tibia were 1.5° and 1.2° in the anterior-posterior (AP) and lateral planes respectively, compared to 2.8° and 3.1° in the Zenith group. This difference reached significance (p=<0.05) using the Mann-Whitney U test. At 2 years, MOx-FQ scores had fallen from pre-operative mean of 63.9∓17.1 to 15∓12.7. EQ-5D VAS scores had improved from 71.3∓17.3 to 81.4∓9.7 points. Radiation exposure had a mean screening time of 82∓29.4s and a decrease in exposure per patient was observed over time. No patients have undergone, or are awaiting, revision surgery. Complications include one intraoperative medial malleolar tip avulsion fracture, one medial malleolar stress fracture, and one patient who developed CRPS.

Conclusions: We present evidence that this system achieves better anatomical alignment of the components when compared to techniques without fluoroscopic navigation. The implant survival and complication profile at a minimum of two years is satisfactory.
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http://dx.doi.org/10.1016/j.fas.2018.11.007DOI Listing
December 2019

Air Pollution Exposure in Walking School Bus Routes: A New Zealand Case Study.

Int J Environ Res Public Health 2018 12 10;15(12). Epub 2018 Dec 10.

Research and Investigations, Auckland Council, Auckland 1010, New Zealand.

Walking School Buses (WSBs), organized groups for children to walk to school under the supervision of adults, help reduce traffic congestion and contribute towards exercise. Routes are based largely on need, traffic safety and travel time, with exposure to air pollution not generally considered. This paper explores whether reductions in exposure can be achieved based on the side of the road travelled using data collected in Auckland, New Zealand. Exposure to air pollution was measured for a 25-min commute consisting of a 10-min segment along a quiet cul-de-sac and a 15-min segment along a main arterial road with traffic congestion heavier in one direction. Two participants were each equipped with a portable P-Trak ultrafine particle monitor and a portable Langan carbon monoxide monitor, and walked the route on opposite sides of the road simultaneously, for both morning and afternoon, logging 10-s data. The results suggest that pedestrians travelling on the footpath next to the less congested side of the road in the morning avoid many short-term peaks in concentration and experience significantly lower mean exposures than those travelling on the footpath next to the more congested side. Significant reductions in air pollution exposure could be made for children by taking into account the side of the road in WSB route design.
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http://dx.doi.org/10.3390/ijerph15122802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313482PMC
December 2018

Conidial Morphogenesis and Septin-Mediated Plant Infection Require Smo1, a Ras GTPase-Activating Protein in .

Genetics 2019 01 16;211(1):151-167. Epub 2018 Nov 16.

School of Biosciences, University of Exeter, EX4 4QD, UK

The pathogenic life cycle of the rice blast fungus involves a series of morphogenetic changes, essential for its ability to cause disease. The mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in , including conidia, appressoria, and asci. All attempts to clone the gene by map-based cloning or complementation have failed over many years. Here, we report the identification of by a combination of bulk segregant analysis and comparative genome analysis. encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.
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http://dx.doi.org/10.1534/genetics.118.301490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325701PMC
January 2019

Osmotrophy.

Curr Biol 2018 10;28(20):R1179-R1180

Biosciences, Living Systems Institute, University of Exeter, Exeter, UK.

What do you do when your food is too big to chew, or worse, when you don't have a stomach at all? Richards and Talbot explain how osmotrophs get around the problem by digesting on the outside.
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http://dx.doi.org/10.1016/j.cub.2018.07.069DOI Listing
October 2018

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.

Sci Rep 2018 09 25;8(1):14355. Epub 2018 Sep 25.

School of Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope Building, Exeter, EX4 4QD, UK.

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.
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http://dx.doi.org/10.1038/s41598-018-32702-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156577PMC
September 2018

Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates.

Phytopathology 2019 Apr 4;109(4):504-508. Epub 2019 Mar 4.

1 Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.
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http://dx.doi.org/10.1094/PHYTO-06-18-0199-LEDOI Listing
April 2019

An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants.

Curr Biol 2018 07 21;28(13):2136-2144.e7. Epub 2018 Jun 21.

Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK; Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK. Electronic address:

Cell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.
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http://dx.doi.org/10.1016/j.cub.2018.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041470PMC
July 2018

MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae.

Autophagy 2018 31;14(9):1543-1561. Epub 2018 Aug 31.

a State Key Laboratory of Hybrid Rice, Key Laboratory of Major Crop Diseases & Collaborative Innovation Center for Hybrid Rice in Yangtze River Basin, Rice Research Institute , Sichuan Agricultural University , Chengdu , China.

Autophagy is essential for appressorium-mediated plant infection by Magnaporthe oryzae, the causal agent of rice blast disease and a major threat to global food security. The regulatory mechanism of pathogenicity-associated autophagy, however, remains largely unknown. Here, we report the identification and functional characterization of a plausible ortholog of yeast SNT2 in M. oryzae, which we term MoSNT2. Deletion mutants of MoSNT2 are compromised in autophagy homeostasis and display severe defects in autophagy-dependent fungal cell death and pathogenicity. These mutants are also impaired in infection structure development, conidiation, oxidative stress tolerance and cell wall integrity. MoSnt2 recognizes histone H3 acetylation through its PHD1 domain and thereby recruits the histone deacetylase complex, resulting in deacetylation of H3. MoSnt2 binds to promoters of autophagy genes MoATG6, 15, 16, and 22 to regulate their expression. In addition, MoTor controls MoSNT2 expression to regulate MoTor signaling which leads to autophagy and rice infection. Our study provides evidence of a direct link between MoSnt2 and MoTor signaling and defines a novel epigenetic mechanism by which MoSNT2 regulates infection-associated autophagy and plant infection by the rice blast fungus.

Abbreviations: M. oryzae: Magnaporthe oryzae; S. cerevisiae: Saccharomyces cerevisiae; F. oxysporum: Fusarium oxysporum; U. maydis: Ustilago maydis; Compl.: complemented strains of ΔMosnt2 expressing MoSNT2-GFP; ATG: autophagy-related; HDAC: histone deacetylase complex; Tor: target of rapamycin kinase; MTOR: mechanistic target of rapamycin kinase in mammals; MoSnt2: DNA binding SaNT domain protein in M. oryzae; MoTor: target of rapamycin kinase in M. oryzae; MoAtg8: autophagy-related protein 8 in M. oryzae; MoHos2: hda one similar protein in M. oryzae; MoeIf4G: eukaryotic translation initiation factor 4 G in M. oryzae; MoRs2: ribosomal protein S2 in M. oryzae; MoRs3: ribosomal protein S3 in M. oryzae; MoIcl1: isocitrate lyase in M. oryzae; MoSet1: histone H3K4 methyltransferase in M. oryzae; Asd4: ascus development 4; Abl1: AMP-activated protein kinase β subunit-like protein; Tig1: TBL1-like gene required for invasive growth; Rpd3: reduced potassium dependency; KAT8: lysine (K) acetyltransferase 8; PHD: plant homeodomain; ELM2: Egl-27 and MTA1 homology 2; GFP: green fluorescent protein; YFP: yellow fluorescent protein; YFP: C-terminal fragment of YFP; YFP: N-terminal fragment of YFP; GST: glutathione S-transferase; bp: base pairs; DEGs: differentially expressed genes; CM: complete medium; MM-N: minimum medium minus nitrogen; CFW: calcofluor white; CR: congo red; DAPI: 4', 6-diamidino-2-phenylindole; BiFC: bimolecular fluorescence complementation; RT: reverse transcription; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RNAi: RNA interference; ChIP: chromatin immunoprecipitation.
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http://dx.doi.org/10.1080/15548627.2018.1458171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135590PMC
October 2019