BackgroundThere is growing interest in the role of DNA methylation in regulating the transcriptio... more BackgroundThere is growing interest in the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in brain disorders characterized by mitochondrial dysfunction. Here, we present a novel approach to interrogate the mitochondrial DNA methylome at single base resolution using targeted bisulfite sequencing. We applied this method to investigate mitochondrial DNA methylation patterns in post-mortem superior temporal gyrus and cerebellum brain tissue from seven human donors.ResultsWe show that mitochondrial DNA methylation patterns are relatively low but conserved, with peaks in DNA methylation at several sites, such as within the D-LOOP and the genes MT-ND2, MT-ATP6, MT-ND4, MT-ND5 and MT-ND6, predominantly in a non-CpG context. The elevated DNA methylation we observe in the D-LOOP we validate using pyrosequencing. We identify loci that show differential DNA methylation patterns associated with age, sex and brain region. Finally, we replicate previou...
Targeted bisulfite sequencing analysis of candidate genes associated with Alzheimer’s disease
Alzheimer's & Dementia, 2021
The histopathological changes in Alzheimer’s disease (AD), including extensive deposits of amyloi... more The histopathological changes in Alzheimer’s disease (AD), including extensive deposits of amyloid β plaques and neurofibrillary tangles occur many years before the onset of clinical symptoms and epigenetic processes such as DNA methylation may contribute to this delay. Recent epigenome‐wide association studies (EWAS) have identified a number of loci in specific genes that show robust and reproducible alterations in DNA methylation changes in AD brain samples. However, the technologies used for these studies only assess a limited number of methylation sites in each gene and further analysis of methylation changes across the entire gene are required to determine the exact extent and pattern of methylation changes in disease. In this study we have performed targeted bisulfite sequencing and RNA sequencing in the Brains for Dementia Research (BDR) tissue sample resource, which is a highly characterised cohort containing tissue with a high degree of standardised pathological, clinical a...
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a si... more 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. In recent years, the use of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided Cas9 endonuclease, has facilitated genome editing technologies and become the leading tool used to generate specific changes to DNA sequences in a wide range of species 1. To generate double stranded breaks (DSBs) in the genome of a target organism, the CRISPR-Cas9 system requires Cas9 endonuclease, which cleaves target DNA at a genomic target sequence 2-4 , and a single RNA molecule, which in the CRISPR-Cas9 system, uses a linker sequence to join the nuclease-binding tracrRNA and the target specific crRNA molecules found in naturally occurring complexes in the source organism, Streptococcus pyogenes 5. The sgRNA associates with the nuclease and directs it to a genomic target sequence 5. The DSB created by the nuclease can then be repaired by non-homologous DNA-end joining (NHEJ) or using homologous recombination (HR), by introduction of donor DNA homologous to the sequence around the break, which allows very specific edits to the DNA sequence, or very precise insertions or deletions 6. The only target sequence requirement necessary for CRISPR-Cas9 genome editing is the presence of the protospacer adjacent motif (PAM), a triplet NGG located immediately 3′ of the genomic target sequence 7,8. Because HR-based repair can be used to introduce modifications at some distance to the DSB, for example up to 30 bp in human stem cells 9 , the majority of fungal genomes are accessible to manipulations using CRISPR-Cas9 editing. CRISPR-Cas9 genome editing offers huge potential to accelerate the pace of research in key fungal research areas, such as biotechnology, medical mycology and plant pathology, by dramatically reducing the time required to undertake common objectives, such as targeted gene deletion, overexpression, single nucleotide changes, or tagging the products of genes of interest with fluorescent proteins 10. The technique also permits targeting of gene families, making multiple mutations 11 , and generating mutants in dikaryotic, or polyploid fungi 12. Moreover, the potential exists to carry out 'selectable marker-free' manipulations for precise genetic changes, a prerequisite for
Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we ... more Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.
Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae
To cause rice blast disease the filamentous fungus Magnaporthe oryzae secretes a battery of effec... more To cause rice blast disease the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signalling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls e...
Cogeme Phytopathogenic Fungi and Oomycete EST Database
Expressed sequence tags (ESTs) have been obtained from eighteen species of plant pathogenic fungi... more Expressed sequence tags (ESTs) have been obtained from eighteen species of plant pathogenic fungi, two species of phytopathogenic oomycete and three species of saprophytic fungi. Hierarchical clustering software was used to classify together ESTs representing the same gene and produce a single contig, or consensus sequence. The unisequence set for each pathogen therefore represents a set of unique gene sequences, each one consisting of either a single EST or a contig sequence made from a group of ESTs. Unisequences were annotated based on top hits against the NCBI non-redundant protein database using blastx.BBSRCThe Cogeme README file contains important information about the set up and use of the dataset
ABSTRACTWheat, the most important food crop, is threatened by a blast disease pandemic. Here, we ... more ABSTRACTWheat, the most important food crop, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America, and to guide pre-emptive wheat breeding for blast resistance.
ice blast disease is an important threat to global food security 1. The disease starts when asexu... more ice blast disease is an important threat to global food security 1. The disease starts when asexual spores of Magnaporthe oryzae, called conidia, land on the hydrophobic surface of a rice leaf inducing differentiation of an infection cell called an appressorium 1-3. The appressorium develops turgor of up to 8.0 MPa due to glycerol accumulation, which generates osmotic pressure 4. Glycerol is maintained in the appressorium by melanin in the cell wall, which reduces its porosity 4,5. Development of the appressorium is tightly linked to the cell cycle, autophagy 6-8 and metabolic checkpoint control mediated by TOR kinase and the cAMP-dependent protein kinase A (PKA) pathway 9-11. Appressorium turgor is monitored by a sensor kinase, Sln1, and once a threshold is reached 12 , septin GTPases in the appressorium pore form a hetero-oligomeric complex that scaffolds cortical F-actin at the base of the appressorium 13,14. This leads to force generation to pierce the cuticle with a rigid penetration hypha. Once inside the leaf, invasive hyphae colonize the first epidermal cell before seeking out plasmodesmata-rich pit fields through which the fungus invades neighbouring cells 15. M. oryzae actively suppresses plant immunity using fungal effector proteins delivered into plant cells 16. After five days, disease lesions appear from which the fungus sporulates to colonize neighbouring plants. Formation of an appressorium by M. oryzae requires a conserved pathogenicity mitogen-activated protein kinase (MAPK), called Pmk1 (ref. 17). Pmk1 mutants cannot form appressoria or cause plant infection, even when plants are wounded 17. Instead, Δpmk1 mutants produce undifferentiated germlings that undergo several rounds of mitosis and septation 17,18. Pmk1 is also responsible for lipid and glycogen mobilization to the appressorium, autophagy in the conidium 4,8,19,20 and invasive cell-to-cell movement 15. A set of pl surface sensors 21 that trigger cAMP-PKA signalling are required for Pmk1 activation 17 , and a TOR-dependent nutrient sensing pathway is necessary for appressorium formation, acting upstream, or perhaps independently, of Pmk1 (refs. 9-11). The mechanism by which Pmk1 exerts such an important role in plant infection has remained largely unknown and only one transcriptional regulator, Mst12, which may act downstream of Pmk1, has been characterized in detail. Mst12 mutants form appressoria normally but are non-functional and cannot cause disease 22. In this study we set out to identify the mechanism by which major transcriptional changes are regulated during appressorium development by M. oryzae. We identified major temporal changes in gene expression in response to an appressorium-inductive hydrophobic Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network
Nicholas J (2019) A sensor kinase controls turgor-driven plant infection by the rice blast fungus... more Nicholas J (2019) A sensor kinase controls turgor-driven plant infection by the rice blast fungus. Nature, 574. pp. 423-427.
The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morpho... more The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 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. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskel...
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a si... more 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 i...
Blast disease destroys up to 30% of the rice crop annually and threatens global food security. Th... more Blast disease destroys up to 30% of the rice crop annually and threatens global food security. The blast fungus invades plant tissue with hyphae that proliferate and grow from cell to cell, often through pit fields, where plasmodesmata cluster. We showed that chemical genetic inhibition of a single fungal mitogen-activated protein (MAP) kinase, Pmk1, prevents from infecting adjacent plant cells, leaving the fungus trapped within a single plant cell. Pmk1 regulates expression of secreted fungal effector proteins implicated in suppression of host immune defenses, preventing reactive oxygen species generation and excessive callose deposition at plasmodesmata. Furthermore, Pmk1 controls the hyphal constriction required for fungal growth from one rice cell to the neighboring cell, enabling host tissue colonization and blast disease.
Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein l... more Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.
The gene content of a plant pathogenic fungus Within the M. grisea genome, 11,109 genes were pred... more The gene content of a plant pathogenic fungus Within the M. grisea genome, 11,109 genes were predicted with articles
The rice blast fungus Magnaporthe grisea develops specialized infection structures known as appre... more The rice blast fungus Magnaporthe grisea develops specialized infection structures known as appressoria, which develop enormous turgor pressure to bring about plant infection. Turgor is generated by accumulation of compatible solutes, including glycerol, which is synthesized in large quantities in the appressorium. Glycogen, trehalose and lipids represent the most abundant storage products in M. grisea conidia. Trehalose and glycogen are rapidly degraded during conidial germination and it is known that trehalose synthesis is required for virulence of the fungus. Lipid bodies are transported to the developing appressoria and degraded at the onset of turgor generation, in a process that is cAMP-dependent. A combined biochemical and genetic approach is being used to dissect the process of turgor generation in the rice blast fungus.
The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of spec... more 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 ...
Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy... more Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. Fungi successfully coupled osmotrophy with filamentous growth, similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete 'pseudofungus'; <i>Hyphochytrium catenoides</i>. Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration acro...
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