Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.

Long-Read Genome Sequence Resource of Ascochyta versabilis Causing Leaf Spot Disease in Pseudostellaria heterophylla.

Ascochyta versabilis is the fungal pathogen that causes the severe leaf spot disease of Pseudostellaria heterophylla (Miq.) Pax, a vital Chinese herbal plant. Here, we deployed PacBio single-molecule real-time long-read sequencing technology to generate a near-complete genome assembly for the A. versabilis KC1 strain and obtained a total of 9.80 Gb raw reads. These reads were processed into a 41.05 Mb genome assembly containing 95 contigs with N50 of 1.70 Mb and a maximum length of 3.93 Mb. A total of 10,457 gene models, of which 1,004 encode putatively secreted proteins, were identified in the genome. This high-quality genome assembly and gene annotation resource will facilitate the institution of functional genetic studies aimed at providing a better insight into the infection mechanisms of A. versabilis to support the development of effective control strategies for leaf spot disease of P. heterophylla.

Lectins from the Edible Mushroom Agaricus bisporus and Their Therapeutic Potentials.

The mushroom Agaricus bisporus secretes biologically active compounds and proteins with benefits for human health. Most reported proteins from A. bisporus are tyrosinases and lectins. Lectins are of therapeutic or pharmaceutical interest. To date, only limited information is available on A. bisporus lectins and lectin-like proteins. No therapeutic products derived from A. bisporus lectin (ABL) are available on the market despite its extensive exploration. Recently, A. bisporus mannose-binding protein (Abmb) was discovered. Its discovery enriches the information and increases the interest in proteins with therapeutic potential from this mushroom. Furthermore, the A. bisporus genome reveals the possible occurrence of other lectins in this mushroom that may also have therapeutic potential. Most of these putative lectins belong to the same lectin groups as ABL and Abmb. Their relationship is discussed. Particular attention is addressed to ABL and Abmb, which have been explored for their potential in medicinal or pharmaceutical applications. ABL and Abmb have anti-proliferative activities toward cancer cells and a stimulatory effect on the immune system. Possible scenarios for their use in therapy and modification are also presented.

Whole-genome and time-course dual RNA-Seq analyses reveal chronic pathogenicity-related gene dynamics in the ginseng rusty root rot pathogen Ilyonectria robusta.

Ilyonectria robusta causes rusty root rot, the most devastating chronic disease of ginseng. Here, we for the first time report the high-quality genome of the I. robusta strain CD-56. Time-course (36 h, 72 h, and 144 h) dual RNA-Seq analysis of the infection process was performed, and many genes, including candidate effectors, were found to be associated with the progression and success of infection. The gene expression profile of CD-56 showed a trend of initial inhibition and then gradually returned to a profile similar to that of the control. Analyses of the gene expression patterns and functions of pathogenicity-related genes, especially candidate effector genes, indicated that the stress response changed to an adaptive response during the infection process. For ginseng, gene expression patterns were highly related to physiological conditions. Specifically, the results showed that ginseng defenses were activated by CD-56 infection and persisted for at least 144 h thereafter but that the mechanisms invoked were not effective in preventing CD-56 growth. Moreover, CD-56 did not appear to fully suppress plant defenses, even in late stages after infection. Our results provide new insight into the chronic pathogenesis of CD-56 and the comprehensive and complex inducible defense responses of ginseng root to I. robusta infection.

Genome Resource for Two Stemphylium vesicarium Isolates Causing Stemphylium Leaf Blight of Onion in New York.

Stemphylium leaf blight caused by Stemphylium vesicarium was recently identified as an emerging disease and dominant in the foliar disease complex affecting onion in New York. Here, we report the genomes of two isolates of S. vesicarium, On16-63 and On16-391. The availability of the genomes will accelerate genomic studies of S. vesicarium, including population biology, sexual reproduction, and fungicide resistance. Additionally, comparative genomics with the other published genome of S. vesicarium causing brown spot of pear will help understand pathogen biology and underpin the development of management strategies for this disease.

Genome Sequence Resource for Ilyonectria mors-panacis, Causing Rusty Root Rot of Panax notoginseng.

Ilyonectria mors-panacis is the cause of a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, antifatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

Whole Genome Sequences of the Tea Leaf Spot Pathogen Didymella segeticola.

The fungal pathogen Didymella segeticola (basionym Phoma segeticola) causes leaf spot on tea (Camellia sinensis), which leads to a loss in tea leaf production in Guizhou Province, China. D. segeticola isolate GZSQ-4 was sequenced using Illumina HiSeq and Pacific Biosciences (PacBio) RS technologies, and then assembled to approximately 33.4 Mbp with a scaffold N50 value of approximately 2.3 Mbp. In total, 10,893 genes were predicted using the Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. The whole-genome sequence of D. segeticola will provide a resource for future research on host-pathogen interactions, determination of trait-specific genes, pathogen evolution, and plant-host adaptation mechanisms.

Genome-wide sequencing and metabolic annotation of Pythium irregulare CBS 494.86: understanding Eicosapentaenoic acid production.

BACKGROUND: Pythium irregulare is an oleaginous Oomycete able to accumulate large amounts of lipids, including Eicosapentaenoic acid (EPA). EPA is an important and expensive dietary supplement with a promising and very competitive market, which is dependent on fish-oil extraction. This has prompted several research groups to study biotechnological routes to obtain specific fatty acids rather than a mixture of various lipids. Moreover, microorganisms can use low cost carbon sources for lipid production, thus reducing production costs. Previous studies have highlighted the production of EPA by P. irregulare, exploiting diverse low cost carbon sources that are produced in large amounts, such as vinasse, glycerol, and food wastewater. However, there is still a lack of knowledge about its biosynthetic pathways, because no functional annotation of any Pythium sp. exists yet. The goal of this work was to identify key genes and pathways related to EPA biosynthesis, in P. irregulare CBS 494.86, by sequencing and performing an unprecedented annotation of its genome, considering the possibility of using wastewater as a carbon source. RESULTS: Genome sequencing provided 17,727 candidate genes, with 3809 of them associated with enzyme code and 945 with membrane transporter proteins. The functional annotation was compared with curated information of oleaginous organisms, understanding amino acids and fatty acids production, and consumption of carbon and nitrogen sources, present in the wastewater. The main features include the presence of genes related to the consumption of several sugars and candidate genes of unsaturated fatty acids production. CONCLUSIONS: The whole metabolic genome presented, which is an unprecedented reconstruction of P. irregulare CBS 494.86, shows its potential to produce value-added products, in special EPA, for food and pharmaceutical industries, moreover it infers metabolic capabilities of the microorganism by incorporating information obtained from literature and genomic data, supplying information of great importance to future work.

Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Genome sequence of the cauliflower mushroom Sparassis crispa (Hanabiratake) and its association with beneficial usage.

Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and ß-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential ß-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of ß-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast ß-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae.

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

Linking secondary metabolites to biosynthesis genes in the fungal endophyte Cyanodermella asteris: The anti-cancer bisanthraquinone skyrin.

Fungal aromatic polyketides display a very diverse and widespread group of natural products. Due to their excellent light absorption properties and widely studied biological activities, they offer numerous application for food, textile and pharmaceutical industry. The biosynthetic pathways of fungal aromatic polyketides usually involve a set of successive enzymes, in which a non-reductive polyketide synthase iteratively catalyzes the essential assembly of simple building blocks into (often polycyclic) aromatic compounds. However, only a limited number of such pathways have been described so far and further elucidation of the individual biosynthetic steps is needed to fully exploit the biotechnological and medicinal potential of these compounds. Here, we identified the bisanthraquinone skyrin as the main pigment of the fungus Cyanodermella asteris, an endophyte that has recently been isolated from the traditional Chinese medicinal plant Aster tataricus. The genome of C. asteris was sequenced, assembled and annotated, which enables first insights into a genome from a non-lichenized member of the class Lecanoromycetes. Genetic and in silico analyses led to the identification of a gene cluster of five genes suggested to encode the enzymatic pathway for skyrin. Our study is a starting point for rational pathway engineering in order to drive the production towards higher yields or more active derivatives. Moreover, our investigations revealed a large potential of secondary metabolite production in C. asteris as well as in all Lecanoromycetes of which genomes were available. These findings convincingly emphasize that Lecanoromycetes are prolific producers of secondary metabolites.

Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3.

The basidiomycetes fungus Rhizoctonia solani AG3 is responsible for black scurf disease on potato and occurs in each potato growing area world-wide. In this study, the draft genome sequence of the black scurf pathogen R. solani AG3-PT isolate Ben3 is presented. The genome sequence of R. solani AG3-PT isolate Ben3 consists of 1385 scaffolds. These scaffolds amount to a size of approx. 51 Mb. Considering coverage analyses of contigs, the size of the diploid genome was estimated to correspond to 116 Mb. Gene prediction by applying AUGUSTUS (3.2.1.) resulted in 12,567 identified genes. Based on automatic annotation using GenDBE, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the R. solani AG3-PT isolate Ben3 genome. Comparative analyses including the R. solani AG3 isolate Rhs1AP, also originating from potato, revealed first insights into core genes shared by both isolates and unique determinants of each isolate.

Draft genome sequence of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB strain BBA69670.

Rhizoctonia solani is a widespread plant pathogenic fungus featuring a broad host range including several economically important crops. Accordingly, genome analyses of R. solani isolates are important to uncover their pathogenic potential. Draft genome sequences for four R. solani isolates representing three of the 14 R. solani anastomosis groups (AGs) are available. Here, we present the first draft genome sequence for an R. solani AG2-2IIIB isolate that is pathogenic on sugar beet. The fungal genome was assembled in 2065 scaffolds consisting of 5826 contigs amounting to a size of about 52 Mb which is larger than any other R. solani isolate known today. Genes potentially encoding cellulolytic, lignolytic and pectinolytic enzymes were identified.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

Genome and transcriptome analysis of the basidiomycetous yeast Pseudozyma antarctica producing extracellular glycolipids, mannosylerythritol lipids.

Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.

Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin.

BACKGROUND: Pectins are diverse and very complex biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. RESULTS: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. CONCLUSIONS: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.

Genome sequence of the model medicinal mushroom Ganoderma lucidum.

Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi.

Genome-based bioprospecting of microbes for new therapeutics.

Bioprospecting of natural sources for new medicines has a long and successful history, exemplified by the fact that over 50% of all drugs currently on the market are either derived from or inspired by natural products. However, development of new natural product-based therapeutics has been on the decline over the past 20 years, mainly owing to frequent re-discovery of already known compounds coupled with high costs for screening, characterization and development. With the onset of the genomic era allowing rapid sequencing and analysis of bacterial and fungal genomes, it became evident that these organisms possess 'hidden treasures' in the form of gene clusters potentially governing biosynthesis of novel biologically active compounds. This review highlights current progress in mining for and expression of these gene clusters, which may revolutionize the drug discovery pipelines in the near future.

Lactic-acid stress causes vacuolar fragmentation and impairs intracellular amino-acid homeostasis in Saccharomyces cerevisiae.

To gain more insight into adaptation response to lactic-acid stress in yeast, a genome-wide screening for genes whose disruption caused hypersensitivity to 4.0% l-lactic acid (pH 2.8) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 107 genes that contributed significantly to the ability of yeast cells to adapt lactic-acid stress. More than 30% of the genes identified in this screening were newly identified to be involved in mechanisms for adaptation response to lactic acid. We found that protein urmylation by Uba4 and N-terminal acetylation by Nat3 were involved in lactic acid adaptation mechanisms. Functional categorization of the genes followed by microscopic analysis revealed that a variety of cellular functions were involved in adaptation response to lactic acid and function associated with vacuolar transport played important roles in adaptation response to lactic acid. We also found that vacuole fragmented immediately upon exposure to lactic- and hydrochloric-acid stress. In addition, our analysis revealed that lactic-acid stress significantly reduced the amount of intracellular amino acids. Amino acid supplementation recovered the adaptation deficiency to lactic acid, suggesting that intracellular amino-acid homeostasis plays important roles in adaptation response to lactic-acid stress. These data suggest that enhancing vacuolar integrity, as well as maintaining intracellular amino-acid homeostasis may be an efficient approach to confer resistance to lactic-acid stress.