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September 22, 2019

Genomic insights into the non-histamine production and proteolytic and lipolytic activities of Tetragenococcus halophilus KUD23.

Tetragenococcus halophilus KUD23, a non-histamine producer, was isolated from a traditional Korean high-salt fermented soybean paste, doenjang. The strain was safe in terms of antibiotic susceptibility, hemolytic activity and biofilm formation. It could grow on De Man-Rogosa-Sharpe agar containing 21% (w/v) NaCl, exhibited acid production at 15% NaCl, and had strain-specific proteolytic and lipolytic activities under salt stress. Complete genome analysis of T. halophilus KUD23 and comparative genomic analysis shed light on the genetic background behind these phenotypic characteristics, including non-production of histamine and proteolytic and lipolytic activities.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 22, 2019

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms.

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44?Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41?Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.

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September 22, 2019

Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains.

Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains.We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains.We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

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September 22, 2019

Genome sequence of the Japanese oak silk moth, Antheraea yamamai: the first draft genome in the family Saturniidae.

Antheraea yamamai, also known as the Japanese oak silk moth, is a wild species of silk moth. Silk produced by A. yamamai, referred to as tensan silk, shows different characteristics such as thickness, compressive elasticity, and chemical resistance compared with common silk produced from the domesticated silkworm, Bombyx mori. Its unique characteristics have led to its use in many research fields including biotechnology and medical science, and the scientific as well as economic importance of the wild silk moth continues to gradually increase. However, no genomic information for the wild silk moth, including A. yamamai, is currently available.In order to construct the A. yamamai genome, a total of 147G base pairs using Illumina and Pacbio sequencing platforms were generated, providing 210-fold coverage based on the 700-Mb estimated genome size of A. yamamai. The assembled genome of A. yamamai was 656 Mb (>2 kb) with 3675 scaffolds, and the N50 length of assembly was 739 Kb with a 34.07% GC ratio. Identified repeat elements covered 37.33% of the total genome, and the completeness of the constructed genome assembly was estimated to be 96.7% by Benchmarking Universal Single-Copy Orthologs v2 analysis. A total of 15 481 genes were identified using Evidence Modeler based on the gene prediction results obtained from 3 different methods (ab initio, RNA-seq-based, known-gene-based) and manual curation.Here we present the genome sequence of A. yamamai, the first genome sequence of the wild silk moth. These results provide valuable genomic information, which will help enrich our understanding of the molecular mechanisms relating to not only specific phenotypes such as wild silk itself but also the genomic evolution of Saturniidae.© The Authors 2017. Published by Oxford University Press.

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September 22, 2019

Reference assembly and annotation of the Pyrenophora teres f. teres isolate 0-1.

Pyrenophora teres f.teres, the causal agent of net form net blotch (NFNB) of barley, is a destructive pathogen in barley-growing regions throughout the world. Typical yield losses due to NFNB range from 10 to 40%; however, complete loss has been observed on highly susceptible barley lines where environmental conditions favor the pathogen. Currently, genomic resources for this economically important pathogen are limited to a fragmented draft genome assembly and annotation, with limited RNA support of theP. teresf.teresisolate 0-1. This research presents an updated 0-1 reference assembly facilitated by long-read sequencing and scaffolding with the assistance of genetic linkage maps. Additionally, genome annotation was mediated by RNAseq analysis using three infection time points and a pure culture sample, resulting in 11,541 high-confidence gene models. The 0-1 genome assembly and annotation presented here now contains the majority of the repetitive content of the genome. Analysis of the 0-1 genome revealed classic characteristics of a “two-speed” genome, being compartmentalized into GC-equilibrated and AT-rich compartments. The assembly of repetitive AT-rich regions will be important for future investigation of genes known as effectors, which often reside in close proximity to repetitive regions. These effectors are responsible for manipulation of the host defense during infection. This updatedP. teresf.teresisolate 0-1 reference genome assembly and annotation provides a robust resource for the examination of the barley-P. teresf.tereshost-pathogen coevolution. Copyright © 2018 Wyatt et al.

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September 22, 2019

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019

Comparative mapping of the ASTRINGENCY locus controlling fruit astringency in hexaploid persimmon (Diospyros kaki Thunb.) with the diploid D. lotus reference genome

Persimmon (Diospyros kaki) is a tree crop species that originated in East Asia, consists mainly of hexaploid individuals (2n = 6x = 90) with some nonaploid individuals. One of the unique characteristics of persimmon is the continuous accumulation of proanthocyanidins (PAs) in its fruit until the middle of fruit development, resulting in a strong astringent taste even at commercial fruit maturity. Among persimmon cultivars, pollination-constant and non-astringent (PCNA) types cease PA accumulation in early fruit development and become non-astringent at commercial maturity. PCNA is an allelic trait to non-PCNA and is controlled by a single locus called the ASTRINGENCY (AST) locus. Previous segregation analyses indicated that the AST locus shows hexasomic inheritance; a recessive allele, ast, at this locus confers PCNA. Here, we report a shuttle mapping approach to delimit the AST locus region in the hexaploid persimmon genome by using D. lotus, a diploid relative of D. kaki, as a reference. A D. lotus F1 population of 333 individuals and 296 D. kaki siblings segregating for the PCNA trait were used to map the AST region using haplotype-specific markers covering the AST region. This indicated that the AST locus is syntenic to an approximately 915-kb region of the D. lotus genome. In this 915-kb region, we found several candidates for AST that were revealed from the fruit transcriptome of a population segregating for the PCNA trait. These results could provide important clues for the isolation of AST in hexaploid persimmon.

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September 22, 2019

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2? diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

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September 22, 2019

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology.Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery.The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

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September 22, 2019

Adaptive strategies of Bacillus thuringiensis isolated from acid mine drainage site in Sabah, Malaysia.

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

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September 22, 2019

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

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September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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September 22, 2019

Complete genome sequence of Pseudomonas Parafulva PRS09-11288, a biocontrol strain produces the antibiotic phenazine-1-carboxylic acid.

Rhizoctonia solani is a plant pathogenic fungus, which can infect a wide range of economic crops including rice. In this case, biological control of this pathogen is one of the fundmental way to effectively control this pathogen. The Pseudomonas parafulva strain PRS09-11288 was isolated from rice rhizosphere and shows biocontrol ability against R. solani. Here, we analyzed the P. parafulva genome, which is ~?4.7 Mb, with 4310 coding sequences, 76 tRNAs, and 7 rRNAs. Genome analysis identified a phenazine biosynthetic pathway, which can produce antibiotic phenazine-1-carboxylic acid (PCA). This compound is responsible for biocontrol ability against R. solani Kühn, which is one of the most serious fungus disease on rice. Analysis of the phenazine biosynthesis gene mutant, ?phzF, which is very important in this pathway, confirmed the relationship between the pathway and PCA production using LC-MS profiles. The annotated full genome sequence of this strain sheds light on the role of P. parafulva PRS09-11288 as a biocontrol bacterium.

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