Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling

In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling

A molecular insight into algal-oomycete warfare: cDNA analysis of <i>Ectocarpus siliculosus</i> infected with the basal oomycete <i>Eurychasma dicksonii</i>

Brown algae are the predominant primary producers in coastal habitats, and like land plants are subject to disease and parasitism. Eurychasma dicksonii is an abundant, and probably cosmopolitan, obligate biotrophic oomycete pathogen of marine brown algae. Oomycetes (or water moulds) are pathogenic or saprophytic non-photosynthetic Stramenopiles, mostly known for causing devastating agricultural and aquacultural diseases. Whilst molecular knowledge is restricted to crop pathogens, pathogenic oomycetes actually infect hosts from most eukaryotic lineages. Molecular evidence indicates that Eu. dicksonii belongs to the most early-branching oomycete clade known so far. Therefore Eu. dicksonii is of considerable interest due to its presumed environmental impact and phylogenetic position. Here we report the first large scale functional molecular data acquired on the most basal oomycete to date. 9873 unigenes, totalling over 3.5Mb of sequence data, were produced from Sanger-sequenced and pyrosequenced EST libraries of infected Ectocarpus siliculosus. 6787 unigenes (70%) were of algal origin, and 3086 (30%) oomycete origin. 57% of Eu. dicksonii sequences had no similarity to published sequence data, indicating that this dataset is largely unique. We were unable to positively identify sequences belonging to the RXLR and CRN groups of oomycete effectors identified in higher oomycetes, however we uncovered other unique pathogenicity factors. These included putative algal cell wall degrading enzymes, cell surface proteins, and cyclophilin-like proteins. A first look at the host response to infection has also revealed movement of the host nucleus to the site of infection as well as expression of genes responsible for strengthening the cell wall, and secretion of proteins such as protease inhibitors. We also found evidence of transcriptional reprogramming of E. siliculosus transposable elements and of a viral gene inserted in the host genome

A molecular insight into algal-oomycete warfare : cDNA analysis of Ectocarpus siliculosus infected with the basal oomycete Eurychasma dicksonii

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Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Abstract not availableMarcia Saraiva, Irene de Bruijn, Laura Grenville-Briggs, Debbie McLaggan, Ariane Willems, Vincent Bulone, Pieter van Wes

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

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

Cell entry of a host targeting protein of oomycetes requires gp96

This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.Peer reviewedPublisher PD

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Background: Pythium ultimum (P. ultimum) is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions although surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report in a genome outside the metazoans. Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae

Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato

Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans