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July 7, 2019  |  

Fungal genome and mating system transitions facilitated by chromosomal translocations involving intercentromeric recombination.

Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.

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July 7, 2019  |  

Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors.

Common bacterial blight is a devastating bacterial disease of common bean (Phaseolus vulgaris) caused by Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli. These phylogenetically distant strains are able to cause similar symptoms on common bean, suggesting that they have acquired common genetic determinants of adaptation to common bean. Transcription Activator-Like (TAL) effectors are bacterial type III effectors that are able to induce the expression of host genes to promote infection or resistance. Their capacity to bind to a specific host DNA sequence suggests that they are potential candidates for host adaption.To study the diversity of tal genes from Xanthomonas strains responsible for common bacterial blight of bean, whole genome sequences of 17 strains representing the diversity of X. citri pv. fuscans and X. phaseoli pv. phaseoli were obtained by single molecule real time sequencing. Analysis of these genomes revealed the existence of four tal genes named tal23A, tal20F, tal18G and tal18H, respectively. While tal20F and tal18G were chromosomic, tal23A and tal18H were carried on plasmids and shared between phylogenetically distant strains, therefore suggesting recent horizontal transfers of these genes between X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. Strikingly, tal23A was present in all strains studied, suggesting that it played an important role in adaptation to common bean. In silico predictions of TAL effectors targets in the common bean genome suggested that TAL effectors shared by X. citri pv. fuscans and X. phaseoli pv. phaseoli strains target the promoters of genes of similar functions. This could be a trace of convergent evolution among TAL effectors from different phylogenetic groups, and comforts the hypothesis that TAL effectors have been implied in the adaptation to common bean.Altogether, our results favour a model where plasmidic TAL effectors are able to contribute to host adaptation by being horizontally transferred between distant lineages.

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July 7, 2019  |  

Complete genome sequences of the plant pathogens Ralstonia solanacearum type strain K60 and R. solanacearum race 3 biovar 2 strain UW551.

Ralstonia solanacearum is a globally distributed plant pathogen that causes bacterial wilt diseases of many crop hosts, threatening both sustenance farming and industrial agriculture. Here, we present closed genome sequences for the R. solanacearum type strain, K60, and the cool-tolerant potato brown rot strain R. solanacearum UW551, a highly regulated U.S. select agent pathogen. Copyright © 2017 Hayes et al.

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July 7, 2019  |  

First complete genome sequences of Xanthomonas citri pv. vignicola strains CFBP7111, CFBP7112, and CFBP7113 obtained using long-read technology

Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors. Copyright © 2017 Ruh et al.

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July 7, 2019  |  

Complete genome sequence of Ralstonia solanacearum FJAT-91, a high-virulence pathogen of tomato wilt.

Ralstonia solanacearum FJAT-91, which displays higher virulence toward plants belonging to the family Solanaceae, was isolated from a wilted tomato plant vessel in Fujian province, southeast China. Here, we report the complete genome sequence of R. solanacearum FJAT-91 using long-read single-molecule PacBio sequencing technology. The genome comprises a 3,873,214-bp circular chromosome and a 2,000,873-bp circular megaplasmid with an overall G+C content of 66.85%. Copyright © 2017 Chen et al.

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July 7, 2019  |  

Harnessing whole genome sequencing in medical mycology.

Comparative genome sequencing studies of human fungal pathogens enable identification of genes and variants associated with virulence and drug resistance. This review describes current approaches, resources, and advances in applying whole genome sequencing to study clinically important fungal pathogens.Genomes for some important fungal pathogens were only recently assembled, revealing gene family expansions in many species and extreme gene loss in one obligate species. The scale and scope of species sequenced is rapidly expanding, leveraging technological advances to assemble and annotate genomes with higher precision. By using iteratively improved reference assemblies or those generated de novo for new species, recent studies have compared the sequence of isolates representing populations or clinical cohorts. Whole genome approaches provide the resolution necessary for comparison of closely related isolates, for example, in the analysis of outbreaks or sampled across time within a single host.Genomic analysis of fungal pathogens has enabled both basic research and diagnostic studies. The increased scale of sequencing can be applied across populations, and new metagenomic methods allow direct analysis of complex samples.

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July 7, 2019  |  

Mechanisms of adaptive divergence and speciation in Littorina saxatilis: Integrating knowledge from ecology and genetics with new data emerging from genomic studies

New opportunities to understand marine speciation and evolution of local adaptation come with genomic approaches and with the development of comprehensive model systems. The marine snail Littorina saxatilis is one example of a developing marine model for investigating genetic mechanisms of rapid divergence and evolution in natural systems. This species is strongly polymorphic and shows formation of local ecotypes throughout its distribution. Support is strong for primary (in situ) and parallel formation of reproductively semi-isolated ecotypes with contact zones between heterogeneous intertidal microhabitats. This makes this species an ideal organism for gaining new insights into the interplay of divergent selection, gene flow and genetic drift during local adaptation and speciation. A relatively well-resolved draft genome and a genetic map describing 17 linkage groups (“chromosomes”) are key tools for investigating the role of structural genomic variation, such as inversions, gene duplications and translocations. Whole genome re-sequencing of pools of individuals and the first comprehensive study of a contact zone contribute direct information on selection and barriers to gene flow present in specific regions of the genome. Linking selection at the phenotypic level to patterns obser ved in the genome is under way by quantitative trait loci mapping and annotation of candidate genes, while the role of single mutations on individual fitness will have to await development of gene manipulation tools. The features of the snail system facilitate the study of local adaptation and speciation and its genomic basis, but the underlying evolutionary processes are expected to be similar in other organisms, and hence this species is a useful model.

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July 7, 2019  |  

Glaucophyta

The Glaucophyta is by far the least species-rich phylum of the Archaeplastida comprising only four described genera, Glaucocystis, Cyanophora, Gloeochaete, and Cyanoptyche, and 15 species. However, recent molecular and morphological analyses reveal that glaucophytes are not as species poor as hitherto assumed with many novel lineages existing in natural environments. Glaucophytes are freshwater phototrophs of moderate to low abundance and retain many ancestral plastid traits derived from the cyanobacterial donor of this organelle, including the remnant peptidoglycan wall in their envelope. These plastids were originally named “cyanelles,” which was later changed to “muroplasts” when their shared ancestry with other Archaeplastida was recognized. The model glaucophyte, Cyanophora paradoxa, is well studied with respect to biochemistry, proteomics, and the gene content of the nuclear and organelle genomes. Investigation of the biosynthesis of cytosolic starch led to a model for the transition from glycogen to starch storage during plastid endosymbiosis. The photosynthetic apparatus, including phycobilisome antennae, resembles that of cyanobacteria. However, the carbon-concentrating mechanism is algal in nature and based on pyrenoids. Studies on protein import into muroplasts revealed a primordial Toc/Tic translocon. The peptidoglycan wall was elucidated with respect to composition, biosynthesis, and involvement of nuclear genes. The muroplast genome is distinct, not due to the number of encoded genes but, rather, because of the presence of unique genes not present on other plastid genomes. The mosaic nature of the gene-rich (27,000) nuclear genome came as a surprise, considering the relatively small genomes of unicellular red algae.

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July 7, 2019  |  

Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex3, similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019  |  

Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens.

The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available.A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) effector genes, which are phylogenetically distant from previous known TAL effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL effector-specific wheat gene expression modulation.PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL effectors of XT4699 strain play roles in modulating wheat host gene expressions.

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July 7, 2019  |  

Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: A randomized trial in children from Bangladesh

Background Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. Method T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. Findings No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. Interpretation Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage–bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. Funding The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.

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July 7, 2019  |  

In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

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July 7, 2019  |  

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain’s virulence.

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July 7, 2019  |  

Resurgence of less-studied smut fungi as models of phytopathogenesis in the -omics era.

The smut fungi form a large, diverse, and non-monophyletic group of plant pathogens that have long served as both important pests of human agriculture but also as fertile organisms of scientific investigation. As modern techniques of molecular genetic analysis became available, many previously-studied species that proved refractive to these techniques fell by the wayside to become neglected. Now, as the advent of rapid and affordable next-generation sequencing provides genomic and transcriptomic resources for even these “forgotten” fungi, several species are making a come-back and retaking prominent places in phytopathogenic research. In this review, we highlight several of these smut fungi, with special emphasis on Microbotryum lychnidis-dioicae, an anther smut, whose molecular genetic tools have finally begun to catch up with its historical importance in classical genetics and now provide mechanistic insights for ecological studies, evolution of host/pathogen interaction, and investigations of emerging infectious disease.

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