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Growing amounts of genomic data and more efficient assembly tools advance organelle genomics at an unprecedented scale. Genomic resources are increasingly used for phylogenetic analyses of many plant species, but are less frequently used to investigate within-species variability and phylogeography. In this study, we investigated genetic diversity of Fagus sylvatica, an important broadleaved tree species of European forests, based on complete chloroplast genomes of 18 individuals sampled widely across the species distribution. Our results confirm the hypothesis of a low cpDNA diversity in European beech. The chloroplast genome size was remarkably stable (158,428 ± 37 bp). The polymorphic markers, 12 microsatellites (SSR), four SNPs and one indel, were found only in the single copy regions, while inverted repeat regions were monomorphic both in terms of length and sequence, suggesting highly efficient suppression of mutation. The within-individual analysis of polymorphisms showed >9k of markers which were proportionally present in gene and non-gene areas. However, an investigation of the frequency of alternate alleles revealed that the source of this diversity originated likely from nuclear-encoded plastome remnants (NUPTs). Phylogeographic and Mantel correlation analysis based on the complete chloroplast genomes exhibited clustering of individuals according to geographic distance in the first distance class, suggesting that the novel markers and in particular the cpSSRs could provide a more detailed picture of beech population structure in Central Europe.
The constitution and regulation of effector repertoires shape host–microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.
Obligate endoparasitic oomycetes are known to ubiquitously occur in marine and freshwater diatoms, but their diversity is still largely unexplored. Many of these parasitoids are members of the early-diverging oomycete lineages (Miracula, Diatomophthora), others are within the Leptomitales of the Saprolegniomycetes (Ectrogella, Lagenisma) and some have been described in the Peronosporomycetes (Aphanomycopsis, Lagenidium). Even though some species have been recently described and two new genera were introduced (Miracula and Diatomophthora), the phylogeny and taxonomy of most of these organisms remain unresolved. This is contrasted by the high number of sequences from unclassified species, as recently revealed from environmental sequencing, suggesting the presence of several undiscovered species. In this study, a new species of Miracula is reported from a marine centric diatom (Minidiscus sp.) isolated from Skagaströnd harbor in Northwest Iceland. The morphology and life cycle traits of this novel oomycete parasite are described herein, and its taxonomic placement within the genus Miracula is confirmed by molecular phylogeny. As it cannot be assigned to any previously described species, it is introduced as Miracula islandica in this study. The genus Miracula thus contains three described holocarpic species (M. helgolandica, M. islandica, M. moenusica) to which likely additional species will need to be added in the future, considering the presence of several lineages known only from environmental sequencing that clustered within the Miracula clade.
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Chloroplasts are difficult to assemble because of the presence of large inverted repeats. At the same time, correct assemblies are important, as chloroplast loci are frequently used for biogeography and population genetics studies. In an attempt to elucidate the orientation of the single-copy regions and to find suitable loci for chloroplast single nucleotide polymorphism (SNP)-based studies, circular chloroplast sequences for the ultra-centenary reference individual of European Beech (Fagus sylvatica), Bhaga, and an additional Polish individual (named Jamy) was obtained based on hybrid assemblies. The chloroplast genome of Bhaga was 158,458 bp, and that of Jamy was 158,462 bp long. Using long-read mapping on the configuration inferred in this study and the one suggested in a previous study, we found an inverted orientation of the small single-copy region. The chloroplast genome of Bhaga and of the individual from Poland both have only two mismatches as well as three and two indels as compared to the previously published genome, respectively. The low divergence suggests low seed dispersal but high pollen dispersal. However, once chloroplast genomes become available from Pleistocene refugia, where a high degree of variation has been reported, they might prove useful for tracing the migration history of Fagus sylvatica in the Holocene.
Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host–pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex.
The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize–U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize–U. maydis interaction.
Similar to chloroplast loci, mitochondrial markers are frequently used for genotyping, phylogenetic studies, and population genetics, as they are easily amplified due to their multiple copies per cell. In a recent study, it was revealed that the chloroplast offers little variation for this purpose in central European populations of beech. Thus, it was the aim of this study to elucidate, if mitochondrial sequences might offer an alternative, or whether they are similarly conserved in central Europe. For this purpose, a circular mitochondrial genome sequence from the more than 300-year-old beech reference individual Bhaga from the German National Park Kellerwald-Edersee was assembled using long and short reads and compared to an individual from the Jamy Nature Reserve in Poland and a recently published mitochondrial genome from eastern Germany. The mitochondrial genome of Bhaga was 504,730 bp, while the mitochondrial genomes of the other two individuals were 15 bases shorter, due to seven indel locations, with four having more bases in Bhaga and three locations having one base less in Bhaga. In addition, 19 SNP locations were found, none of which were inside genes. In these SNP locations, 17 bases were different in Bhaga, as compared to the other two genomes, while 2 SNP locations had the same base in Bhaga and the Polish individual. While these figures are slightly higher than for the chloroplast genome, the comparison confirms the low degree of genetic divergence in organelle DNA of beech in central Europe, suggesting the colonisation from a common gene pool after the Weichsel Glaciation. The mitochondrial genome might have limited use for population studies in central Europe, but once mitochondrial genomes from glacial refugia become available, it might be suitable to pinpoint the origin of migration for the re-colonising beech population.
Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.
Diatoms are thought to provide about 40% of total global photosynthesis and diatoms of the genus Coscinodiscus are an important, sometimes dominant, cosmopolitan component of the marine diatom community. The oomycete parasitoid Lagenisma coscinodisci is widespread in the northern hemisphere on its hosts in the genus Coscinodiscus. Because of its potential ecological importance, it would be a suitable pathogen model to investigate plankton/parasite interactions, but the species cannot be cultivated on media without its host, so far. Thus, it was the aim of this study to explore the potential of dual culture of host and pathogen in the laboratory and to optimise cultivation to ensure a long-term cultivation of the pathogen. Here, we report successful cultivation of a single spore strain of L. coscinodisci (Isla), on several Coscinodiscus species and strains, as well as the establishment of a cultivation routine with Coscinodiscus granii (CGS1 and CG36), which enabled us to maintain the single spore strain for more than 3 years in 6 cm Petri dishes and 10 ml tissue culture flasks. This opens up the opportunity to study the processes and mechanism in plankton/parasitoid interactions under controlled conditions.
The oomycete genus Ectrogella currently comprises a rather heterogeneous group of obligate endoparasitoids, mostly of diatoms and algae. Despite their widespread occurrence, little is known regarding the phylogenetic affinities of these bizarre organisms. Traditionally, the genus was included within the Saprolegniales, based on zoospore diplanetism and a saprolegnia/achlya-like zoospore discharge. The genus has undergone multiple re-definitions in the past, and has often been used largely indiscriminately for oomycetes forming sausage-like thalli in diatoms. While the phylogenetic affinity of the polyphyletic genus Olpidiopsis has recently been partially resolved, taxonomic placement of the genus Ectrogella remained unresolved, as no sequence data were available for species of this genus. In this study, we report the phylogenetic placement of Ectrogella bacillariacearum infecting the freshwater diatom Nitzschia sigmoidea. The phylogenetic reconstruction shows that Ectrogella bacillariacearum is grouped among the early diverging lineages of the Saprolegniomycetes with high support, and is unrelated to the monophyletic diatom-infecting olpidiopsis-like species. As these species are neither related to Ectrogella, nor to the early diverging lineages of Olpidiopsis s. str. and Miracula, they are placed in a new genus, Diatomophthora, in the present study.
Holocarpic oomycetes are poorly known but widespread parasites in freshwater and marine ecosystems. Most of the holocarpic species seem to belong to clades that diverge before the two crown lineages of the oomycetes, the Saprolegniomycetes and the Peronosporomycetes. Recently, the genus Miracula was described to accommodate Miracula helgolandica, a holocarpic parasitoid of Pseudo-nitzschia diatoms, which received varying support for its placement as the earliest-diverging oomycete lineage. In the same phylogenetic reconstruction, Miracula helgolandica was grouped with some somewhat divergent sequences derived from environmental sequencing, indicating that Miracula would not remain monotypic. Here, a second species of Miracula is reported, which was found as a parasitoid in the limnic centric diatom Pleurosira leavis. Its life-cycle stages are described and depicted in this study and its phylogenetic placement in the genus Miracula revealed. As a consequence, the newly discovered species is introduced as Miracula moenusica.
Olpidiopsis is a genus of obligate holocarpic endobiotic oomycetes. Most of the species classified in the genus are known only from their morphology and life cycle, and a few have been examined for their ultrastructure or molecular phylogeny. However, the taxonomic placement of all sequenced species is provisional, as no sequence data are available for the type species, O. saprolegniae, to consolidate the taxonomy of species currently placed in the genus. Thus, efforts were undertaken to isolate O. saprolegniae from its type host, Saprolegnia parasitica and to infer its phylogenetic placement based on 18S rDNA sequences. As most species of Olpidiopsis for which sequence data are available are from rhodophyte hosts, we have also isolated the type species of the rhodophyte-parasitic genus Pontisma, P. lagenidioides and obtained partial 18S rDNA sequences. Phylogenetic reconstructions in the current study revealed that O. saprolegniae from Saprolegnia parasitica forms a monophyletic group with a morphologically similar isolate from S. ferax, and a morphologically and phylogenetically more divergent species from S. terrestris. However, they were widely separated from a monophyletic, yet unsupported clade containing P. lagenidioides and red algal parasites previously classified in Olpidiopsis. Consequently, all holocarpic parasites in red algae should be considered to be members of the genus Pontisma as previously suggested by some researchers. In addition, a new species of Olpidiopsis, O. parthenogenetica is introduced to accommodate the pathogen of S. terrestris.
Background: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.
Results: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.
Conclusions: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
Background: Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Results: Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Conclusions: Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.
Ceraceosorus bombacis is an early-diverging lineage of smut fungi and a pathogen of cotton trees (Bombax ceiba). To study the evolutionary genomics of smut fungi in comparison with other fungal and oomycete pathogens, the genome of C. bombacis was sequenced and comparative genomic analyses were performed. The genome of 26.09 Mb encodes for 8,024 proteins, of which 576 are putative-secreted effector proteins (PSEPs). Orthology analysis revealed 30 ortholog PSEPs among six Ustilaginomycotina genomes, the largest groups of which are lytic enzymes, such as aspartic peptidase and glycoside hydrolase. Positive selection analyses revealed the highest percentage of positively selected PSEPs in C. bombacis compared with other Ustilaginomycotina genomes. Metabolic pathway analyses revealed the absence of genes encoding for nitrite and nitrate reductase in the genome of the human skin pathogen Malassezia globosa, but these enzymes are present in the sequenced plant pathogens in smut fungi. Interestingly, these genes are also absent in cultivable oomycete animal pathogens, while nitrate reductase has been lost in cultivable oomycete plant pathogens. Similar patterns were also observed for obligate biotrophic and hemi-biotrophic fungal and oomycete pathogens. Furthermore, it was found that both fungal and oomycete animal pathogen genomes are lacking cutinases and pectinesterases. Overall, these findings highlight the parallel evolution of certain genomic traits, revealing potential common evolutionary trajectories among fungal and oomycete pathogens, shaping the pathogen genomes according to their lifestyle.
Even though the microevolution of plant hosts and pathogens has been intensely studied, knowledge regarding macro-evolutionary patterns is limited. Having the highest species diversity and host-specificity among Oomycetes, downy mildews are a useful a model for investigating long-term host-pathogen coevolution. We show that phylogenies of Bremia and Asteraceae are significantly congruent. The accepted hypothesis is that pathogens have diverged contemporarily with their hosts. But maximum clade age estimation and sequence divergence comparison reveal that congruence is not due to long-term coevolution but rather due to host-shift driven speciation (pseudo-cospeciation). This pattern results from parasite radiation in related hosts, long after radiation and speciation of the hosts. As large host shifts free pathogens from hosts with effector triggered immunity subsequent radiation and diversification in related hosts with similar innate immunity may follow, resulting in a pattern mimicking true co-divergence, which is probably limited to the terminal nodes in many pathogen groups.
Background: Xanthophyllomyces dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. dendrorhous for a phylogenomic investigation of the Basidiomycota.
Results: The genome assembly of a haploid strain of Xanthophyllomyces dendrorhous revealed a genome of 19.50 Megabases with 6385 protein coding genes. Phylogenetic analyses were conducted including 48 fungal genomes. These revealed Ustilaginomycotina and Agaricomycotina as sister groups. In the latter a well-supported sister-group relationship of two major orders, Polyporales and Russulales, was inferred. Wallemia occupies a basal position within the Agaricomycotina and X. dendrorhous represents the basal lineage of the Tremellomycetes, highlighting that the typical tremelloid parenthesomes have either convergently evolved in Wallemia and the Tremellomycetes, or were lost in the Cystofilobasidiales lineage. A detailed characterization of the CoA-related pathways was done and all genes for fatty acid, sterol and carotenoid synthesis have been assigned.
Conclusions: The current study ascertains that Wallemia with tremelloid parenthesomes is the most basal agaricomycotinous lineage and that Cystofilobasidiales without tremelloid parenthesomes are deeply rooted within Tremellomycetes, suggesting that parenthesomes at septal pores might be the core synapomorphy for the Agaricomycotina. Apart from evolutionary insights the genome sequence of X. dendrorhous will facilitate genetic pathway engineering for optimized astaxanthin or oxidative alcohol production.
Pseudoperonospora cubensis, an obligate biotrophic oomycete causing devastating foliar disease in species of the Cucurbitaceae family, was never reported in seeds or transmitted by seeds. We now show that P. cubensis occurs in fruits and seeds of downy mildew-infected plants but not in fruits or seeds of healthy plants. About 6.7% of the fruits collected during 2012–2014 have developed downy mildew when homogenized and inoculated onto detached leaves and 0.9% of the seeds collected developed downy mildew when grown to the seedling stage. This is the first report showing that P. cubensis has become seed-transmitted in cucurbits. Species-specific PCR assays showed that P. cubensis occurs in ovaries, fruit seed cavity and seed embryos of cucurbits. We propose that international trade of fruits or seeds of cucurbits might be associated with the recent global change in the population structure of P. cubensis.