Fungal Genome Sequences Research Articles

Genomics-Driven Discovery of Phytotoxic Cytochalasans Involved in the Virulence of the Wheat Pathogen Parastagonospora nodorum.

The etiology of fungal pathogenesis of grains is critical to global food security. The large number of orphan biosynthetic gene clusters uncovered in fungal plant pathogen genome sequencing projects suggests that we have a significant knowledge gap about the secondary metabolite repertoires of these pathogens and their roles in plant pathogenesis. Cytochalasans are a family of natural products of significant interest due to their ability to bind to actin and interfere with cellular processes that involved actin polymerization; however, our understanding of their biosynthesis and biological roles remains incomplete. Here, we identified a putative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster (phm) that was upregulated in the pathogen Parastagonospora nodorum during its infection on wheat. Overexpression of the transcription factor gene phmR encoded in the phm gene cluster resulted in the production of two leucine-derived cytochalasans, phomacins D and E (1 and 2, respectively), and an acetonyl adduct phomacin F. Heterologous expression of the PKS-NRPS gene phmA and the trans-enoyl reductase (ER) gene phmE in Aspergillus nidulans resulted in the production of a novel 2-pyrrolidone precursor prephomacin. Reverse genetics and wheat seedling infection assays showed that ΔphmA mutants exhibited significantly reduced virulence compared to the wild type. We further demonstrated that both 1 and 2 showed potent actin polymerization-inhibitory activities and exhibited potentially monocot-specific antigerminative activities. The findings from this study have advanced our knowledge based on the biosynthesis and biological roles of cytochalasans, the latter of which could have significant implications for our understanding of the molecular mechanisms of fungus-plant interactions.

AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline.

Secondary metabolites produced by bacteria and fungi are an important source of antimicrobials and other bioactive compounds. In recent years, genome mining has seen broad applications in identifying and characterizing new compounds as well as in metabolic engineering. Since 2011, the ‘antibiotics and secondary metabolite analysis shell—antiSMASH’ (https://antismash.secondarymetabolites.org) has assisted researchers in this, both as a web server and a standalone tool. It has established itself as the most widely used tool for identifying and analysing biosynthetic gene clusters (BGCs) in bacterial and fungal genome sequences. Here, we present an entirely redesigned and extended version 5 of antiSMASH. antiSMASH 5 adds detection rules for clusters encoding the biosynthesis of acyl-amino acids, β-lactones, fungal RiPPs, RaS-RiPPs, polybrominated diphenyl ethers, C-nucleosides, PPY-like ketones and lipolanthines. For type II polyketide synthase-encoding gene clusters, antiSMASH 5 now offers more detailed predictions. The HTML output visualization has been redesigned to improve the navigation and visual representation of annotations. We have again improved the runtime of analysis steps, making it possible to deliver comprehensive annotations for bacterial genomes within a few minutes. A new output file in the standard JavaScript object notation (JSON) format is aimed at downstream tools that process antiSMASH results programmatically.

Marcel van der Heijden.

Marcel van der Heijden.

Understanding photoreception in fungi and its role in fungal development with focus on phytopathogenic fungi

Like other environmental factors, light can influence various developmental processes in fungal species. Most of the fungi can respond to different wavelengths of light such as blue (475 nm), near UV (300–380 nm) and red light (650 nm). Blue light has been found to be the most associated light quality with fungal development. The most commonly investigated photoregulatory receptors in fungi are the white collar complex proteins (WC-1, WC-2) in the light oxygen voltage domain which sense and regulate the effects of blue light on fungal development and reproduction. The sequencing of several fungal genomes has led to the identification of other photoreceptor genes and their products, such as phytochromes (for red light), cryptochromes (for blue light) and blue and green light absorbing rhodopsins in some ascomycetes and basidiomycetes. Although similar types of regulatory mechanisms appear to be involved in signalling light responses in fungal species, nevertheless, there may be other hitherto undiscovered mechanisms responsible for regulating light perception. It warrants further investigations on photoreception in more fungal species including plant pathogenic fungi to better understand the underlying mechanisms controlling light perception at the metabolic level. Certain circadian rhythms have been found to play crucial role in fungal perception and adaptation to light. The information has led to the use of certain photoselective materials for control of some plant diseases.

Introduction: Overview of Fungal Genomics.

Genome sequences and postgenomic tools have had a major impact on fungal research. When the first fungal genome sequences became available it became clear how much more complex fungal biology was than had been previously assumed. Since then an increasing number of genomes have become available enabling detailed comparative studies, especially when combined with postgenomic tools such as transcriptomics and proteomics. This chapter provides an overview and current state of fungal genomics.

Properties Of Two Novel Esterases Identified From Culture Supernatant Of Penicillium Purpurogenum Grown On Sugar Beet Pulp

The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level for the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.

Fungal Genomes and Insights into the Evolution of the Kingdom.

The kingdom Fungi comprises species that inhabit nearly all ecosystems. Fungi exist as both free-living and symbiotic unicellular and multicellular organisms with diverse morphologies. The genomes of fungi encode genes that enable them to thrive in diverse environments, invade plant and animal cells, and participate in nutrient cycling in terrestrial and aquatic ecosystems. The continuously expanding databases of fungal genome sequences have been generated by individual and large-scale efforts such as Génolevures, Broad Institute's Fungal Genome Initiative, and the 1000 Fungal Genomes Project (http://1000.fungalgenomes.org). These efforts have produced a catalog of fungal genes and genomic organization. The genomic datasets can be utilized to better understand how fungi have adapted to their lifestyles and ecological niches. Large datasets of fungal genomic and transcriptomic data have enabled the use of novel methodologies and improved the study of fungal evolution from a molecular sequence perspective. Combined with microscopes, petri dishes, and woodland forays, genome sequencing supports bioinformatics and comparative genomics approaches as important tools in the study of the biology and evolution of fungi.

FunGAP: Fungal Genome Annotation Pipeline using evidence-based gene model evaluation.

Successful genome analysis depends on the quality of gene prediction. Although fungal genome sequencing and assembly have become trivial, its annotation procedure has not been standardized yet. FunGAP predicts protein-coding genes in a fungal genome assembly. To attain high-quality gene models, this program runs multiple gene predictors, evaluates all predicted genes, and assembles gene models that are highly supported by homology to known sequences. To do this, we built a scoring function to estimate the congruency of each gene model based on known protein or domain homology. FunGAP is written in Python script and is available in GitHub ( https://github.com/CompSynBioLab-KoreaUniv/FunGAP ). This software is freely available only for noncommercial users. igchoi@korea.ac.kr. Supplementary data are available at Bioinformatics online.

BIOTECHNOLOGY FOR DIVERSIFICATION AND IMPROVED RESILIENCE OF THE OIL PALM

Oil palm is one of the most important crops in Malaysia and has brought about significant economic and social development to the country. Nevertheless, there are major drawbacks and challenges faced by the industry such as shrinking availability of arable land, labour issues, and pest and disease. This has forced the industry to be more proactive by increasing yield and venturing into synthesis of higher value products. Besides conventional breeding which has made a lot of contributions, biotechnology has been identified as one of the tools to overcome the mentioned challenges. Oil palm biotechnology, especially genetic modification, began more than three decades ago leveraging multidisciplinary research including biochemistry of lipid biosynthesis, isolation of useful genes and promoters and finally genetic transformation. The main target of MPOB’s oil palm genetic modification effort is to produce palms with higher oleic acid content. Other targets are synthesising palmitoleic acid and ricinoleic acid, increasing stearic acid and lycopene (carotenoid) content and production of biodegradable plastics. Biotechnological research also targets towards better understanding the pathogen biology and disease epidemiology for implementing disease management programmes. In the area of disease management, especially for Ganoderma species, study on plant pathogenesis and plant-microbe interaction is also being carried out. Significant achievements have resulted from biochemical studies, isolation and characterisation of important oil palm genes and promoters. Furthermore, transformation of constructs with various targetted products into oil palm embryogenic calli was successfully carried out while the regeneration of transgenic oil palm harbouring the useful genes is in progress. Towards developing oil palm with pest and disease tolerance, efforts have been initiated to understand the oil palm defence mechanisms. Several differentially expressed defence-related genes governing the interaction of the plant pathogen and oil palm have been identified. Besides understanding the oil palm system, research into the pathogen Ganoderma including identification of pathogen strains, developing various markers and data mining of fungal genome sequences have been conducted. Keywords: Genetic modification, promoters, lipid biosynthesis,carotenoid, Ganoderma.

Functional specialization of two paralogous TAF12 variants by their selective association with SAGA and TFIID transcriptional regulatory complexes

TATA box-binding protein (TBP)-associated factors (TAFs), evolutionarily conserved from yeast to humans, play a central role during transcription initiation. A subset of TAF proteins is shared in transcription factor II D (TFIID) and SAGA transcription regulatory complexes. Although higher eukaryotes contain multiple TAF variants that specify tissue- and developmental stage-specific organization of TFIID or SAGA complexes, in unicellular genomes, however, each TAF is encoded by a single gene. Surprisingly, we found that the genome of Candida albicans, the predominant human fungal pathogen, contains two paralogous TAF12 genes, CaTAF12L and CaTAF12, encoding H2B-like histone-fold domain-containing variants. Of the available fungal genome sequences, only seven other closely related diploid pathogenic Candida genomes encode the two TAF12 paralogs. Using affinity purifications from C. albicans cell extracts, we demonstrate that CaTAF12L uniquely associates with the SAGA complex and CaTAF12 associates with the TFIID complex. We further show that CaTAF12, but not CaTAF12L, is essential for C. albicans growth. Conditional depletion of the two TAF12 variant proteins caused distinct cellular and colony phenotypes. Together our results define a specialized organization of the TAF12 variants and non-redundant roles for the two TAF12 variants in the unicellular C. albicans genome.

A plant pathology perspective of fungal genome sequencing

The majority of plant pathogens are fungi and many of these adversely affect food security. This minireview aims to provide an analysis of the plant pathogenic fungi for which genome sequences are publicaNy available, to assess their general genome characteristics, and to consider how genomics has impacted plant pathology. A list of sequenced fungal species was assembled, the taxonomy of all species verified, and the potential reason for sequencing each of the species considered. The genomes of 1090 fungal species are currently (October 2016) in the public domain and this number is rapidly rising. Pathogenic species comprised the largest category (35.5 %) and, amongst these, plant pathogens are predominant. Of the 191 plant pathogenic fungal species with available genomes, 61.3 % cause diseases on food crops, more than half of which are staple crops. The genomes of plant pathogens are slightly larger than those of other fungal species sequenced to date and they contain fewer coding sequences in relation to their genome size. Both of these factors can be attributed to the expansion of repeat elements. Sequenced genomes of plant pathogens provide blueprints from which potential virulence factors were identified and from which genes associated with different pathogenic strategies could be predicted. Genome sequences have also made it possible to evaluate adaptability of pathogen genomes and genomic regions that experience selection pressures. Some genomic patterns, however, remain poorly understood and plant pathogen genomes alone are not sufficient to unravel complex pathogen-host interactions. Genomes, therefore, cannot replace experimental studies that can be complex and tedious. Ultimately, the most promising application lies in using fungal plant pathogen genomics to inform disease management and risk assessment strategies. This will ultimately minimize the risks of future disease outbreaks and assist in preparation for emerging pathogen outbreaks.

Mycotoxins: A Fungal Genomics Perspective.

The chemical and enzymatic diversity in the fungal kingdom is staggering. Large-scale fungal genome sequencing projects are generating a massive catalog of secondary metabolite biosynthetic genes and pathways. Fungal natural products are a boon and bane to man as valuable pharmaceuticals and harmful toxins. Understanding how these chemicals are synthesized will aid the development of new strategies to limit mycotoxin contamination of food and feeds as well as expand drug discovery programs. A survey of work focused on the fumonisin family of mycotoxins highlights technological advances and provides a blueprint for future studies of other fungal natural products. Expressed sequence tags led to the discovery of new fumonisin genes (FUM) and hinted at a role for alternatively spliced transcripts in regulation. Phylogenetic studies of FUM genes uncovered a complex evolutionary history of the FUM cluster, as well as fungi with the potential to synthesize fumonisin or fumonisin-like chemicals. The application of new technologies (e.g., CRISPR) could substantially impact future efforts to harness fungal resources.

Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus.

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multi-modular PKSs and NRPSs; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several novel isoquinoline alkaloids, the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi.

Fungi Contribute Critical but Spatially Varying Roles in Nitrogen and Carbon Cycling in Acid Mine Drainage.

The ecosystem roles of fungi have been extensively studied by targeting one organism and/or biological process at a time, but the full metabolic potential of fungi has rarely been captured in an environmental context. We hypothesized that fungal genome sequences could be assembled directly from the environment using metagenomics and that transcriptomics and proteomics could simultaneously reveal metabolic differentiation across habitats. We reconstructed the near-complete 27 Mbp genome of a filamentous fungus, Acidomyces richmondensis, and evaluated transcript and protein expression in floating and streamer biofilms from an acid mine drainage (AMD) system. A. richmondensis transcripts involved in denitrification and in the degradation of complex carbon sources (including cellulose) were up-regulated in floating biofilms, whereas central carbon metabolism and stress-related transcripts were significantly up-regulated in streamer biofilms. These findings suggest that the biofilm niches are distinguished by distinct carbon and nitrogen resource utilization, oxygen availability, and environmental challenges. An isolated A. richmondensis strain from this environment was used to validate the metagenomics-derived genome and confirm nitrous oxide production at pH 1. Overall, our analyses defined mechanisms of fungal adaptation and identified a functional shift related to different roles in carbon and nitrogen turnover for the same species of fungi growing in closely located but distinct biofilm niches.

RNA silencing approaches for identifying pathogenicity and virulence elements towards engineering crop resistance to plant pathogenic fungi.

Abstract Over the past several years, RNA interference (RNAi) has emerged as a powerful genetic tool that is being exploited not only in fundamental research for the assessment of gene functions, but also in various fields of applied research in medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content, but also for plant protection. In the study of plant-fungus interactions, a comprehensive understanding of pathogenic mechanisms based on information from genome sequences demands advanced genomics tools and infrastructure. In order to make use of the plethora of information yielded by the rapidly increasing fungal pathogen genome sequencing studies, there is a growing need to achieve gene function assessment amenable to high-throughput techniques. Targeted gene disruption is effective but may not be feasible in certain fungi because of lack of transformation protocols or their life styles. RNAi techniques offer an alternative but, in contrast to other biological systems, only few studies have reported the use of RNAi to explore gene function in plant pathogenic fungi. In this review, we summarize a variety of options using RNAi techniques that have become available to study gene function for high-throughput phenotypic screening of virulence and pathogenicity factors in plant fungi. The identification of such genes can be exploited for the protection of crops against fungal pathogens.

FastQFS – A tool for evaluating and filtering paired-end sequencing data generated from high throughput sequencing

Next generation sequencing (NGS) technologies generate huge amounts of sequencing data. Several microbial genome projects, in particular for fungal whole genome sequencing, have used NGS techniques because of their cost efficiency. However, NGS techniques also require computational tools able to process and analyze huge datasets. Data processing steps, including quality and length filters, often lead to a remarkable improvement in the accuracy and quality of data analyses. Choosing appropriate parameters for this purpose is not always straightforward, as these will vary with the dataset. In this study we present the FastQFS (Fastq Quality Filtering and Statistics) tool, which can be used for both an assessment of filtering parameters and read filtering. There are several tools available, but an important asset of FastQFS is that it provides the information of filtering parameters that fit best to the raw dataset, prior to computationally expensive filtering. It generates statistics of reads meeting different quality and length thresholds, and also the expected coverage depth of the genome which would be achieved after applying different filtering parameters. Thus, the FastQFS tool will help researchers to make informed decisions on an NGS read filtering parameters, and avoiding time-consuming optimization of filtering criteria after initial analyses. The source code of the tool and related files are available from 10.12761/SGN.2015.4 .

Transposable Elements in Fungi: A Genomic Approach

Transposable elements (TEs) include a wide range of DNA sequences that can change positions in the genome. The accessibility of whole fungal genome sequences and analysis of TEs demonstrate the important role they play in genome evolution of fungi species. TE activity is a primary mechanism for high fitness, plasticity and adaptability in certain species of pathogenic fungi. Some of the deleterious effects of transposons may be due to ectopic recombination among transposons of the same family. To prevent possible damage caused by the transposons, some fungi possess TE-silencing mechanisms, for instance, RIP (Repeat Induced Pont mutation) and RNA silencing. In addition, TEs are efficient molecular markers due to their structure and transposition strategy. Copies or remnants of both class I (retrotransposons) and class II (DNA transposons) have been identified, and the number of distinct mobile elements continues to grow. Such repeated sequences vary in number as well as in size, and comprise 3 to 20% of the sequenced genome of most fungi. In contrast, the plant pathogenic fungus Blumeria graminis has a genome estimated at 174 Mb, and 85% of the genome includes TEs [3]. More than 215 genus-specific TEs were found in Laccaria bicolor in addition to many remaining degenerate copies. Additionally, 40 different TE families were detected in the genome of L. bicolor, and less than 5% nucleotide mutations had accumulated, which suggest that they are recently acquired elements, and possible activities can be inferred for such elements [4]. In general, the most abundant TEs in sequenced genomes of fungi are Gypsy, Copia and Tc1-Mariner (Figure 1).

The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species.

BackgroundAlternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins.DescriptionWe have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform.ConclusionRecent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu.

Phylogeny of type I polyketide synthases (PKSs) in fungal entomopathogens and expression analysis of PKS genes in Beauveria bassiana BCC 2660

Entomopathogenic fungi are able to invade and kill insects. Various secondary metabolites can mediate the interaction of a fungal pathogen with an insect host and also help the fungus compete with other microbes. Here we screened 23 isolates of entomopathogenic fungi for polyketide synthase (PKS) genes and amplified 72 PKS gene fragments using degenerate PCR. We performed a phylogenetic analysis of conserved ketosynthase and acyltransferase regions in these 72 sequences and 72 PKSs identified from four insect fungal genome sequences. The resulting genealogy indicated 47 orthologous groups with 99–100 % bootstrap support, suggesting shared biosynthesis of identical or closely related compounds from different fungi. Three insect-specific groups were identified among the PKSs in reducing clades IIa, IIb, and III, which comprised PKSs from 12, 9, and 30 fungal isolates, respectively. A IIa-IIb pair could be found in seven fungi. Expression analyses revealed that eleven out of twelve PKS genes identified in Beauveria bassiana BCC 2660 were expressed in culture. PKS genes from insect-specific clades IIa and IIb were expressed only in insect-containing medium, while others were expressed only in PDB or in CYB, PDB and SDY. The data suggest the potential production of several polyketides in culture.