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A domain-centric analysis of oomycete plant pathogen genomes reveals unique protein organization (Online First)
Abstract
Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens
... For each of the 15 proteomes, additional processing of the raw HMMER output was performed using a custom python script to resolve overlapping domain issues. The general rule for solving the domain overlapping problem was adopted from previous work (Seidl et al., 2011) as follows: for non-overlapping domains in the given protein the score of −1 was assigned and the domain remained in the protein. ...
Interactions between proteins underlie all aspects of complex biological mechanisms. Therefore, methodologies based on complex network analyses can facilitate identification of promising candidate genes involved in phenotypes of interest and put this information into appropriate contexts. To facilitate discovery and gain additional insights into globally important pathogenic fungi, we have reconstructed computationally inferred interactomes using an interolog and domain-based approach for 15 diverse Ascomycete fungal species, across nine orders, specifically Aspergillus fumigatus, Bipolaris sorokiniana, Blumeria graminis f. sp. hordei, Botrytis cinerea, Colletotrichum gloeosporioides, Colletotrichum graminicola, Fusarium graminearum, Fusarium oxysporum f. sp. lycopersici, Fusarium verticillioides, Leptosphaeria maculans, Magnaporthe oryzae, Saccharomyces cerevisiae, Sclerotinia sclerotiorum, Verticillium dahliae, and Zymoseptoria tritici. Network cartography analysis was associated with functional patterns of annotated genes linked to the disease-causing ability of each pathogen. In addition, for the best annotated organism, namely F. graminearum, the distribution of annotated genes with respect to network structure was profiled using a random walk with restart algorithm, which suggested possible co-location of virulence-related genes in the protein–protein interaction network. In a second ‘use case’ study involving two networks, namely B. cinerea and F. graminearum, previously identified small silencing plant RNAs were mapped to their targets. The F. graminearum phenotypic network analysis implicates eight B. cinerea targets and 35 F. graminearum predicted interacting proteins as prime candidate virulence genes for further testing. All 15 networks have been made accessible for download at www.phi-base.org providing a rich resource for major crop plant pathogens.
... viticolasecretomeisverysimilartothatpredictedintheH.arabi- dopsidisgenome[10].Themostprevalentdomainsidentifiedwere proteinscontainingglycosylhydrolaseandproteasedomains (Table4)inagreementwithpreviousstudiesthathaveshownthese twoclassesofhighlyabundantenzymestobelinkedtopathoge- nicityinoomycetes[8] [65]. ...
... DUFs remain a treasure trove of novel biology waiting to be explored (Bateman et al., 2010; Jaroszewski et al., 2009). They may also likely to play a role in the lifestyle of pathogens and hence can be promising targets for further experimental validation (Seidl et al., 2011; Ohm et al., 2012). In our analysis, out of 200 hypothetical proteins, 85 showed various protein domains. ...
At the time of infection, many of the gene products of completely sequenced organisms yet remain ‘hypothetical’ meaning they remain unsimilar to any previously characterized and may disguise some true virulent factors. Domain scanning provides a means of understanding functional information in these cases, extending facilitated identification of
their virulence factors, proceeding for antimicrobial drug and vaccine design. In developing countries, mortality rate due to Infective endocarditis is accelerating along with retardation in efficiency of pathogen specific drugs. We have re-annotated at domain level and predicted cellular localization of 200 unique and hypothetical proteins obtained by syntenic comparison of Streptococcus gordonii among other strains of similar species for similar infection. The study resulted into 200 unique and hypothetical proteins, of which, domains of 85 proteins are predictable, representing 15 with no similarity with human proteome. Later, 9 proteins with 8 domains predicted to be antimicrobial targets. Further, these can be experimentally validated for drug and vaccine target ability.
... The complete genome of P. ramorum has been sequenced and annotated , and this has added much to the understanding of oomycete evolution and pathogenicity (e.g., Jiang et al. 2008;Kasuga et al. 2012;Seidl et al. 2011;Win et al. 2012). As we report here, the genome sequence also enables bioinformatic tools to separate pathogen transcripts from the host when both have been concurrently sequenced in infected plant tissue. ...
As part of the Western Forest Transcriptome Survey, we used Massively Parallel Sequencing to concurrently sequence the transcriptomes of a generalist invasive pathogen (Phytophthora ramorum) and its most susceptible California Floristic Province native host (tanoak, Notholithocarpus densiflorus) during the course of their interaction. We analyzed 43.5 Mbp of sequence from 4 experimental conditions, of which 18.8 Mbp mapped to the P. ramorum genome sequence. We report on the characterization of the P. ramorum genes found to be active during infection, the assembly of a de novo reference transcriptome for tanoak, and the characterization of tanoak gene regions differentially expressed after infection, including regions associated with pathogenicity and resistance. These findings are an important step in understanding the genetics of interactions of forest hosts and their pathogens. Pathogen introductions can quickly have devastating effects on species and ecosystems, but developing the breeding populations used in traditional forest genetics is a decades-long undertaking. The application of genomic methods to novel host-plant interactions will ultimately allow a much faster response to a present ecological threat.
... The complete genome of P. ramorum has been sequenced and annotated , and this has added much to the understanding of oomycete evolution and pathogenicity (e.g., Jiang et al. 2008;Kasuga et al. 2012;Seidl et al. 2011;Win et al. 2012). As we report here, the genome sequence also enables bioinformatic tools to separate pathogen transcripts from the host when both have been concurrently sequenced in infected plant tissue. ...
Full text available at http://www.treesearch.fs.fed.us/pubs/47907
Emergent diseases are an ever-increasing threat to forests and forest ecosystems and necessitate the development of research tools for species that often may have few pre-existing resources. We sequenced the mRNA expressed by the sudden oak death pathogen Phytophthora ramorum and its most susceptible forest host, tanoak, within the same tissue at two time points after inoculation, and in uninfected tanoak controls. Using the P. ramorum genome to differentiate host and pathogen transcripts, we detected more than 850 P. ramorum transcripts at 5 days post-inoculation and a concurrent upregulation of host genes usually associated with pathogenicity. At 1 day, in contrast, we did not detect pathogen expression or significant enrichment of functional categories of host transcripts relative to controls, highlighting the importance of sequencing depth for in planta studies of host-pathogen interactions. This study highlights processes in molecular host-pathogen interactions in forest trees and provides a first reference transcriptome for tanoak, allowing the preliminary identification of disease-related genes in this study and facilitating future work for this and other members of the family Fagaceae.
... The Phytophthora lineages The genus Phytophthora has been inferred to be paraphyletic with respect to the downy mildews (Cooke et al. 2000; Runge et al. 2011a), which is also corroborated by some species that exhibit intermediate characteristics (Göker et al. 2003; Thines et al. 2007; Thines 2009). However, using the few genomes available so far, this paraphyly could not be demonstrated (Seidl et al. 2011), although high support for this was gained in multigene analyses on a broad taxon sampling in Phytophthora (Runge et al. 2011a). Species of Phytophthora are hemibiotrophic, with the more basal clades 8–10 having a shorter biotrophic phase compared to the members of the crown clades 1–4 (Cooke et al. 2000; Blair et al. 2008). ...
Oomycetes have colonised all continents and oceans in a great variety of habitats and are arguably one of the most successful eukaryotic lineages. This is contrasted by the limited knowledge available for this group in various fields in comparison to other ubiquitous eukaryotes, such as unikont fungi, animals or plants. In this review an overview is given on the evolution and diversification of the oomycetes, with focus on the plant parasitic lineages and aspects of wild pathosystems.
The understanding of molecular mechanisms underlying host-pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.
Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of Ps. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations was improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome, and nearly 55% of the intron-containing genes from Ps. cubensis, had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in Ps. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations, and through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides further evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant pathogenic oomycetes.
All species continuously evolve to adapt to changing environments. The genetic variation that fosters such adaptation is caused by a plethora of mechanisms, including meiotic recombination that generates novel allelic combinations in the progeny of two parental lineages. However, a considerable number of eukaryotic species, including many fungi, do not have an apparent sexual cycle and are consequently thought to be limited in their evolutionary potential. As such organisms are expected to have reduced capability to eliminate deleterious mutations, they are often considered as evolutionary dead ends. However, inspired by recent reports we argue that such organisms can be as persistent as organisms with conventional sexual cycles through the use of other mechanisms, such as genomic rearrangements, to foster adaptation.
The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, and Pythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthora species and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.
Associations between proteins are essential to understand cell biology. While this complex interplay between proteins has been studied in model organisms, it has not yet been described for the oomycete late blight pathogen Phytophthora infestans.
We present an integrative probabilistic functional gene network that provides associations for 37 percent of the predicted P. infestans proteome. Our method unifies available genomic, transcriptomic and comparative genomic data into a single comprehensive network using a Bayesian approach. Enrichment of proteins residing in the same or related subcellular localization validates the biological coherence of our predictions. The network serves as a framework to query existing genomic data using network-based methods, which thus far was not possible in Phytophthora. We used the network to study the set of interacting proteins that are encoded by genes co-expressed during sporulation. This identified potential novel roles for proteins in spore formation through their links to proteins known to be involved in this process such as the phosphatase Cdc14.
The functional association network represents a novel genome-wide data source for P. infestans that also acts as a framework to interrogate other system-wide data. In both capacities it will improve our understanding of the complex biology of P. infestans and related oomycete pathogens.
Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.
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