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pH Signaling in Human Fungal Pathogens: a New Target for Antifungal Strategies

pH Signaling in Human Fungal Pathogens: a New Target for Antifungal Strategies | Plant pathogenic fungi | Scoop.it

Fungi are exposed to broadly fluctuating environmental conditions, to which adaptation is crucial for their survival. An ability to respond to a wide pH range, in particular, allows them to cope with rapid changes in their extracellular settings. PacC/Rim signaling elicits the primary pH response in both model and pathogenic fungi and has been studied in multiple fungal species. In the predominant human pathogenic fungi, namely, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, this pathway is required for many functions associated with pathogenesis and virulence. Aspects of this pathway are fungus specific and do not exist in mammalian cells. In this review, we highlight recent advances in our understanding of PacC/Rim-mediated functions and discuss the growing interest in this cascade and its factors as potential drug targets for antifungal strategies. We focus on both conserved and distinctive features in model and pathogenic fungi, highlighting the specificities of PacC/Rim signaling in C. albicans, A. fumigatus, and C. neoformans. We consider the role of this pathway in fungal virulence, including modulation of the host immune response. Finally, as now recognized for other signaling cascades, we highlight the role of pH in adaptation to antifungal drug pressure. By acting on the PacC/Rim pathway, it may therefore be possible (i) to ensure fungal specificity and to limit the side effects of drugs, (ii) to ensure broad-spectrum efficacy, (iii) to attenuate fungal virulence, (iv) to obtain additive or synergistic effects with existing antifungal drugs through tolerance inhibition, and (v) to slow the emergence of resistant mutants.

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Previously studied in plant pathogens...

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A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism | Plant pathogenic fungi | Scoop.it

As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community.

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Microsporidia: Eukaryotic Intracellular Parasites Shaped by Gene Loss and Horizontal Gene Transfers - Annual Review of Microbiology

Microsporidia: Eukaryotic Intracellular Parasites Shaped by Gene Loss and Horizontal Gene Transfers - Annual Review of Microbiology | Plant pathogenic fungi | Scoop.it
Microsporidia are eukaryotic parasites of many animals that appear to have adapted to an obligate intracellular lifestyle by modifying the morphology and content of their cells. Living inside other cells, they have lost many, or all, metabolic functions, resulting in genomes that are always gene poor and often very small. The minute content of microsporidian genomes led many to assume that these parasites are biochemically static and uninteresting. However, recent studies have demonstrated that these organisms can be surprisingly complex and dynamic. In this review I detail the most significant recent advances in microsporidian genomics and discuss how these have affected our understanding of many biological aspects of these peculiar eukaryotic intracellular pathogens.

Via Francis Martin
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Six novel species of Fusarium from natural ecosystems in Australia - Online First - Springer

Six novel species of Fusarium from natural ecosystems in Australia - Online First - Springer | Plant pathogenic fungi | Scoop.it

Six new species of Fusarium associated with soil and plant hosts from ecosystems of minimal anthropogenic disturbance in Australia are described. Fusarium coicis from Coix gasteenii, F. goolgardi from Xanthorrhoea glauca, F. mundagurra from soil and Mangifera indica, F. newnesensefrom soil, F. tjaetaba from Sorghum interjectum and F. tjaynera from soil, Triodia microstachya,Sorghum interjectum and Sorghum intrans. Morphology and phylogenetic analysis of EF-1α, RPB1and RPB2 sequence data were used to delineate species boundaries. The new species were phylogenetically distributed in the Fusarium sambucinum, F. fujikuroi, and F. chlamydosporumspecies complexes, and two novel species complexes. These six new species have particular phylogeographic significance as not only do they provide further insight into the geographic patterns of Fusarium evolution but also challenge current phylogeographic hypotheses.

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Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen | Plant pathogenic fungi | Scoop.it

Although entomopathogenic fungi and their invertebrate hosts share a >300 million year co-evolutionary history, little is known concerning the biochemical and genetic basis of insect defensive tactics and the countermeasures evolved and evolving by the pathogen to thwart these defenses. Our results provide a molecular mechanism to help explain why some insects are more resistant to broad host-range entomopathogenic fungi. We identify beetle cuticular secretions and a fungal detoxifying enzyme as components of an arms race between insects and the fungal pathogen, suggesting an evolving role for the quinone reductase enzyme as a specific virulence factor for host quinone detoxification. As races have winners and losers, this paper captures a snapshot where the host is leading the race.

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Clonal reproduction in fungi

Clonal reproduction in fungi | Plant pathogenic fungi | Scoop.it

Research over the past two decades shows that both recombination and clonality are likely to contribute to the reproduction of all fungi. This view of fungi is different from the historical and still commonly held view that a large fraction of fungi are exclusively clonal and that some fungi have been exclusively clonal for hundreds of millions of years. Here, we first will consider how these two historical views have changed. Then we will examine the impact on fungal research of the concept of restrained recombination [Tibayrenc M, Ayala FJ (2012) Proc Natl Acad Sci USA 109 (48):E3305–E3313]. Using animal and human pathogenic fungi, we examine extrinsic restraints on recombination associated with bottlenecks in genetic variation caused by geographic dispersal and extrinsic restraints caused by shifts in reproductive mode associated with either disease transmission or hybridization. Using species of the model yeast Saccharomyces and the model filamentous fungus Neurospora, we examine intrinsic restraints on recombination associated with mating systems that range from strictly clonal at one extreme to fully outbreeding at the other and those that lie between, including selfing and inbreeding. We also consider the effect of nomenclature on perception of reproductive mode and a means of comparing the relative impact of clonality and recombination on fungal populations. Last, we consider a recent hypothesis suggesting that fungi thought to have the most severe intrinsic constraints on recombination actually may have the fewest.

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Great review!

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Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments | Plant pathogenic fungi | Scoop.it

Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition.

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Thorough analysis of agar substitutes

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Changes of Nitric Oxide and Its Relationship with H2O2 and Ca2+ in Defense Interactions between Wheat and Puccinia Triticina

Changes of Nitric Oxide and Its Relationship with H2O2 and Ca2+ in Defense Interactions between Wheat and Puccinia Triticina | Plant pathogenic fungi | Scoop.it

In this research, the wheat cultivar 'Lovrin 10' and Puccinia triticina races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium (Ca2+) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide (NO) and monitoring the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in- cell adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response (HR) at 24–72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred at 4 h after inoculation, and fluorescence was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with our previous results, it could be concluded that, Ca2+ and hydrogen peroxide (H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca2+, NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.

 

 


Via Christophe Jacquet
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The role of the microbiome of truffles in aroma formation: a meta-analysis approach

The role of the microbiome of truffles in aroma formation: a meta-analysis approach | Plant pathogenic fungi | Scoop.it

Truffles (Tuber spp.) are ascomycete subterraneous fungi that form ectomycorrhizas in a symbiotic relationship with plant roots. Their fruiting bodies are appreciated for their distinctive aromas which might be partially derived from microbes. Indeed truffle fruiting bodies are colonized by a diverse microbial community made of bacteria, yeasts, guest filamentous fungi and viruses. The aim of this mini-review is double. First the current knowledge on the microbial community composition of truffles has been synthesized to highlight similarities and differences among four truffle species (T. magnatum, T. melanosporum, T. aestivum and T. borchii) at various stages of their life cycle. Second the potential role of the microbiome in truffle aroma formation has been addressed for the same four species. Our results suggest that on one side odorants which are common to many truffle species might be of mixed truffle and microbial origin while on the other side less common odorants might be derived from microbes only. They also highlight that bacteria, the dominant group in the truffle's microbiome, might also be the most important contributors to truffle aromas not only in T. borchii as already demonstrated but also in T. magnatum, T. aestivum and T. melanosporum.

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Frontiers | A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses | Plant Biotic Inte...

Frontiers | A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses | Plant Biotic Inte... | Plant pathogenic fungi | Scoop.it
We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 μM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase) with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA) in response to the mutants versus wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere.
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Glucosinolate-derived isothiocyanates impact mitochondrial function in fungal cells and elicit an oxidative stress response necessary for growth recovery

Glucosinolate-derived isothiocyanates impact mitochondrial function in fungal cells and elicit an oxidative stress response necessary for growth recovery | Plant pathogenic fungi | Scoop.it

Glucosinolates are brassicaceous secondary metabolites that have long been considered as chemical shields against pathogen invasion. Isothiocyanates (ITCs), are glucosinolate-breakdown products that have negative effects on the growth of various fungal species. We explored the mechanism by which ITCs could cause fungal cell death usingAlternaria brassicicola, a specialist Brassica pathogens, as model organism. Exposure of the fungus to ICTs led to a decreased oxygen consumption rate, intracellular accumulation of reactive oxygen species (ROS) and mitochondrial-membrane depolarization. We also found that two major regulators of the response to oxidative stress, i.e., the MAP kinase AbHog1 and the transcription factor AbAP1, were activated in the presence of ICTs. Once activated by ICT-derived ROS, AbAP1 was found to promote the expression of different oxidative-response genes. This response might play a significant role in the protection of the fungus against ICTs as mutants deficient in AbHog1 or AbAP1 were found to be hypersensitive to these metabolites. Moreover, the loss of these genes was accompanied by a significant decrease in aggressiveness on Brassica. We suggest that the robust protection response against ICT-derived oxidative stress might be a key adaptation mechanism for successful infection of host plants by Brassicaceae-specialist necrotrophs like A. brassicicola.

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Blocking primers reduce co-amplification of plant DNA when studying bacterial endophyte communities

Blocking primers reduce co-amplification of plant DNA when studying bacterial endophyte communities | Plant pathogenic fungi | Scoop.it
A blocking primer set based on the technique described by Vestheim and Jarman (2008) was developed to reduce amplification of non-target plant DNA when conducting metagenomic studies on bacterial endophyte communities. Bacterial amplication efficiency was increased 300-fold compared to standard PCR in an Illumina-based study of Sorghastrum nutans leaves.

Via Jean-Michel Ané
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Jean-Michel Ané's curator insight, July 13, 10:12 PM

Wow... That should be very useful.

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Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea | Plant pathogenic fungi | Scoop.it

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression ofbcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing ofbcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

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RNASequel: accurate and repeat tolerant realignment of RNA-seq reads

RNASequel: accurate and repeat tolerant realignment of RNA-seq reads | Plant pathogenic fungi | Scoop.it

RNA-seq is a key technology for understanding the biology of the cell because of its ability to profile transcriptional and post-transcriptional regulation at single nucleotide resolutions. Compared to DNA sequencing alignment algorithms, RNA-seq alignment algorithms have a diminished ability to accurately detect and map base pair substitutions, gaps, discordant pairs and repetitive regions. These shortcomings adversely affect experiments that require a high degree of accuracy, notably the ability to detect RNA editing. We have developed RNASequel, a software package that runs as a post-processing step in conjunction with an RNA-seq aligner and systematically corrects common alignment artifacts. Its key innovations are a two-pass splice junction alignment system that includesde novo splice junctions and the use of an empirically determined estimate of the fragment size distribution when resolving read pairs. We demonstrate that RNASequel produces improved alignments when used in conjunction with STAR or Tophat2 using two simulated datasets. We then show that RNASequel improves the identification of adenosine to inosine RNA editing sites on biological datasets. This software will be useful in applications requiring the accurate identification of variants in RNA sequencing data, the discovery of RNA editing sites and the analysis of alternative splicing.

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Proposals to clarify and enhance the naming of fungi under the International Code of Nomenclature for algae, fungi, and plants

Proposals to clarify and enhance the naming of fungi under the International Code of Nomenclature for algae, fungi, and plants | Plant pathogenic fungi | Scoop.it
Twenty-three proposals to modify the International Code of Nomenclature for algae, fungi, and plantsadopted in 2011 with respect to the provisions for fungi are made, in accordance with the wishes of mycologists expressed at the 10th International Mycological Congress in Bangkok in 2014, and with the support of the International Commission on the Taxonomy of Fungi (ICTF), the votes of which are presented here. The proposals relate to: conditions for epitypification, registration of later typifications, protected lists of names, removal of exemptions for lichen-forming fungi, provision of a diagnosis when describing a new taxon, citation of sanctioned names, avoiding homonyms in other kingdoms, ending preference for sexually typified names, and treatment of conspecific names with the same epithet. These proposals are also being published in Taxon, will be considered by the Nomenclature Committee for Fungi and General Committee on Nomenclature, and voted on at the 19th International Botanical Congress in Shenzhen, China, in 2017.
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A ToxA-like protein from Cochliobolus heterostrophus induces light-dependent leaf necrosis and acts as a virulence factor with host selectivity on maize

A ToxA-like protein from Cochliobolus heterostrophus induces light-dependent leaf necrosis and acts as a virulence factor with host selectivity on maize | Plant pathogenic fungi | Scoop.it

Highlights
• We identified a ToxA-like gene (ChTOXA) from the maize pathogen C. heterostrophus.
• ChToxA protein is structurally similar to PtrToxA despite lacking the RGD motif.
• ChToxA induces light-dependent necrosis in a host-selective manner on maize.
• Deletion of ChTOXA results in reduced virulence on ChToxA-sensitive maize.
• ChToxA homologues exist in both Dothideomycete and Sordariomycete species.

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Maternal effects on tree phenotypes: considering the microbiome

Maternal effects on tree phenotypes: considering the microbiome | Plant pathogenic fungi | Scoop.it

Highlights
• Biotic and abiotic effects on mother plants modulate the phenotype of the seedlings.
• Plant–microbe interactions affect, and are affected by, maternal effects on seedlings.
• Maternal endophytic communities are expected to be important mediators of biotic maternal effects.
• Understanding maternal effects, including fungal endophyte effects, offers an opportunity for tree improvement.

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A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA

A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA | Plant pathogenic fungi | Scoop.it

Mycoviruses generally contain dsRNA genomes but ssRNA and ssDNA examples are known. Mycovirus diversity is increasing, and here we describe a unique example that contains four dsRNA elements nominated Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1). We show for the first time (to our knowledge) that both purified AfuTmV-1 and its dsRNA are infectious for protoplasts and that the virus genome is not conventionally encapsidated and has a unique organization. Separation of the genes encoding the RNA-dependent RNA polymerase enzyme responsible for copying the viral genome and an S-adenosyl methionine-dependent methyltransferase capping enzyme on different dsRNAs is also previously unreported for a mycovirus. AfuTmV-1 appears to be intermediate between dsRNA and positive-strand ssRNA viruses, as well as between encapsidated and capsidless RNA viruses.

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SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum

SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum | Plant pathogenic fungi | Scoop.it

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans. The expression profile ofSnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta. Subsequent characterization of the ΔSnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of theP. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene inAlternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

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Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa

Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa | Plant pathogenic fungi | Scoop.it

Immune systems distinguish “self” from “non-self” to maintain homeostasis and must differentially gate access to allow colonization by potentially beneficial, non-pathogenic microbes. Plant roots grow within extremely diverse soil microbial communities, but assemble a taxonomically limited root-associated microbiome. We grew isogenic Arabidopsis thaliana mutants with altered immune systems in a wild soil and also in recolonization experiments with a synthetic bacterial community. We established that biosynthesis of, and signaling dependent on, the foliar defense phytohormone salicylic acid is required to assemble a normal root microbiome. Salicylic acid modulates colonization of the root by specific bacterial families. Thus, plant immune signaling drives selection from the available microbial communities to sculpt the root microbiome.

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Would be nice to repeat with fungi and oomycetes

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Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing

Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing | Plant pathogenic fungi | Scoop.it

Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, andGloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.

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Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici | Plant pathogenic fungi | Scoop.it

Highlights
• We have constructed Z. tritici ku70 and ku80 null mutants.
• Gene targeting frequency in the ku null strains is greater than 85%.
• Deletion of KU70 and KU80 does not affect in vitro growth or pathogenicity.
• Sulfonylurea resistance was established as a new positive selection marker in Z. tritici.
• Ternary vectors were constructed to enable yeast recombinational cloning in Z. tritici.

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A gene locus for targeted ectopic gene integration in Zymoseptoria tritici

A gene locus for targeted ectopic gene integration in Zymoseptoria tritici | Plant pathogenic fungi | Scoop.it

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. InZymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single “soft-landing” site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1R allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.

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Nice approach.

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One-step synthesis of fluorescent carbon dots for imaging bacterial and fungal cells - Analytical Methods (RSC Publishing)

One-step synthesis of fluorescent carbon dots for imaging bacterial and fungal cells - Analytical Methods (RSC Publishing) | Plant pathogenic fungi | Scoop.it

In this work, fluorescent carbon dots (C-dots) were synthesized using a hydrothermal method with Punica granatum (pomegranate) fruits as precursors, and were then used as probes for imaging of bacterial (Pseudomonas aeruginosa) and fungal (Fusarium avenaceum) cells. The C-dots showed a strong emission at 453 nm when excited at 383 nm. Rapid internalization of the C-dots by these cells was confirmed by two color (green and red) images that were obtained using confocal fluorescence microscopy.

 
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Interesting way to make a microbe stain.  Not sure if it's very specific.

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A large-scale crop protection bioassay data set

A large-scale crop protection bioassay data set | Plant pathogenic fungi | Scoop.it

ChEMBL is a large-scale drug discovery database containing bioactivity information primarily extracted from scientific literature. Due to the medicinal chemistry focus of the journals from which data are extracted, the data are currently of most direct value in the field of human health research. However, many of the scientific use-cases for the current data set are equally applicable in other fields, such as crop protection research: for example, identification of chemical scaffolds active against a particular target or endpoint, the de-convolution of the potential targets of a phenotypic assay, or the potential targets/pathways for safety liabilities. In order to broaden the applicability of the ChEMBL database and allow more widespread use in crop protection research, an extensive data set of bioactivity data of insecticidal, fungicidal and herbicidal compounds and assays was collated and added to the database.

 
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Microbial species delineation using whole genome sequences

Microbial species delineation using whole genome sequences | Plant pathogenic fungi | Scoop.it

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.

Steve Marek's insight:

Need something like this for eukaryotes too.

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