A Relational Database for the Discovery of Genes Encoding Amino Acid Biosynthetic Enzymes in Pathogenic Fungi

Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html) was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogen Blumeria graminis possesses all amino acid biosynthetic pathways found in free-living fungi, such as Saccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms

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

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

e-Fungi: a data resource for comparative analysis of fungal genomes.

BACKGROUND: The number of sequenced fungal genomes is ever increasing, with about 200 genomes already fully sequenced or in progress. Only a small percentage of those genomes have been comprehensively studied, for example using techniques from functional genomics. Comparative analysis has proven to be a useful strategy for enhancing our understanding of evolutionary biology and of the less well understood genomes. However, the data required for these analyses tends to be distributed in various heterogeneous data sources, making systematic comparative studies a cumbersome task. Furthermore, comparative analyses benefit from close integration of derived data sets that cluster genes or organisms in a way that eases the expression of requests that clarify points of similarity or difference between species. DESCRIPTION: To support systematic comparative analyses of fungal genomes we have developed the e-Fungi database, which integrates a variety of data for more than 30 fungal genomes. Publicly available genome data, functional annotations, and pathway information has been integrated into a single data repository and complemented with results of comparative analyses, such as MCL and OrthoMCL cluster analysis, and predictions of signaling proteins and the sub-cellular localisation of proteins. To access the data, a library of analysis tasks is available through a web interface. The analysis tasks are motivated by recent comparative genomics studies, and aim to support the study of evolutionary biology as well as community efforts for improving the annotation of genomes. Web services for each query are also available, enabling the tasks to be incorporated into workflows. CONCLUSION: The e-Fungi database provides fungal biologists with a resource for comparative studies of a large range of fungal genomes. Its analysis library supports the comparative study of genome data, functional annotation, and results of large scale analyses over all the genomes stored in the database. The database is accessible at http://www.e-fungi.org.uk, as is the WSDL for the web services.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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

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 cytoskeleton at the appressorium pore. Smol physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection

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

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

Comparative Genome Analysis Reveals an Absence of Leucine-Rich Repeat Pattern-Recognition Receptor Proteins in the Kingdom Fungi

Background: In plants and animals innate immunity is the first line of defence against attack by microbial pathogens. Specific molecular features of bacteria and fungi are recognised by pattern recognition receptors that have extracellular domains containing leucine rich repeats. Recognition of microbes by these receptors induces defence responses that protect hosts against potential microbial attack. Methodology/Principal Findings: A survey of genome sequences from 101 species, representing a broad cross-section of the eukaryotic phylogenetic tree, reveals an absence of leucine rich repeat-domain containing receptors in the fungal kingdom. Uniquely, however, fungi possess adenylate cyclases that contain distinct leucine rich repeat-domains, which have been demonstrated to act as an alternative means of perceiving the presence of bacteria by at least one fungal species. Interestingly, the morphologically similar osmotrophic oomycetes, which are taxonomically distant members of the stramenopiles, possess pattern recognition receptors with similar domain structures to those found in plants. Conclusions: The absence of pattern recognition receptors suggests that fungi may possess novel classes of patternrecognition receptor, such as the modified adenylate cyclase, or instead rely on secretion of anti-microbial secondary metabolites for protection from microbial attack. The absence of pattern recognition receptors in fungi, coupled with their abundance in oomycetes, suggests this may be a unique characteristic of the fungal kingdom rather than a consequence o

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Genome-wide characterization of mitochondrial DNA methylation in human brain

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 previously reported differentially methylated regions between brain regions from a methylated DNA immunoprecipitation sequencing study.ConclusionsWe have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain samples. Looking to the future this approach could be utilized to investigate the role of mitochondrial epigenetic mechanisms in disorders that display mitochondrial dysfunction

Genome-wide transcriptional profiling of appressorium development by the rice blast fungus Magnaporthe oryzae.

addresses: College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.notes: PMCID: PMC3276559The rice blast fungus Magnaporthe oryzae is one of the most significant pathogens affecting global food security. To cause rice blast disease the fungus elaborates a specialised infection structure called an appressorium. Here, we report genome wide transcriptional profile analysis of appressorium development using next generation sequencing (NGS). We performed both RNA-Seq and High-Throughput SuperSAGE analysis to compare the utility of these procedures for identifying differential gene expression in M. oryzae. We then analysed global patterns of gene expression during appressorium development. We show evidence for large-scale gene expression changes, highlighting the role of autophagy, lipid metabolism and melanin biosynthesis in appressorium differentiation. We reveal the role of the Pmk1 MAP kinase as a key global regulator of appressorium-associated gene expression. We also provide evidence for differential expression of transporter-encoding gene families and specific high level expression of genes involved in quinate uptake and utilization, consistent with pathogen-mediated perturbation of host metabolism during plant infection. When considered together, these data provide a comprehensive high-resolution analysis of gene expression changes associated with cellular differentiation that will provide a key resource for understanding the biology of rice blast disease

Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus

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