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Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens., (© 2021 John Wiley & Sons Ltd.)

Infection with Aphanomyces invadans is a serious fish disease with major global impacts. Despite affecting over 160 fish species, some of the species like the common carp Cyprinus carpio are resistant to A. invadans infection. In the present study, we investigated the transcriptomes of head kidney of common carp experimentally infected with A. invadans. In time course analysis, 5288 genes were found to be differentially expressed (DEGs), of which 731 were involved in 21 immune pathways. The analysis of immune-related DEGs suggested that efficient processing and presentation of A. invadans antigens, enhanced phagocytosis, recognition of pathogen-associated molecular patterns, and increased recruitment of leukocytes to the sites of infection contribute to resistance of common carp against A. invadans. Herein, we provide a systematic understanding of the disease resistance mechanisms in common carp at molecular level as a valuable resource for developing disease management strategies for this devastating fish-pathogenic oomycete., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oomycetes are fungal-like eukaryotes and many of them are pathogens that threaten natural ecosystems and cause huge financial losses for the aqua- and agriculture industry. Amongst them, Aphanomyces invadans causes Epizootic Ulcerative Syndrome (EUS) in fish which can be responsible for up to 100% mortality in aquaculture. As other eukaryotic pathogens, in order to establish and promote an infection, A. invadans secretes proteins, which are predicted to overcome host defence mechanisms and interfere with other processes inside the host. We investigated the role of Lhs1 which is part of an ER-resident complex that generally promotes the translocation of proteins from the cytoplasm into the ER for further processing and secretion. Interestingly, proteomic studies reveal that only a subset of virulence factors are affected by the silencing of AiLhs1 in A. invadans indicating various secretion pathways for different proteins. Importantly, changes in the secretome upon silencing of AiLhs1 significantly reduces the virulence of A. invadans in the infection model Galleriamellonella. Furthermore, we show that AiLhs1 is important for the production of zoospores and their cluster formation. This renders proteins required for protein ER translocation as interesting targets for the potential development of alternative disease control strategies in agri- and aquaculture., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

This study assessed the impact of routine vaccination of Atlantic salmon pre-smolts on gene expression and the possible link to saprolegniosis on Scottish fish farms. Fish were in 4 different groups 1) 'control' - fish without handling or vaccination 2) 'vaccinated' - fish undergoing full vaccination procedure 3) 'non vaccinated' - fish undergoing full vaccination procedure but not vaccinated and 4) 'vaccinated-MH' - fish undergoing vaccination, but procedure involved minimal handling. A strong increase in cortisol and glucose levels was observed after 1 h in all groups relative to the control group. Only in the non-vaccinated group did the level decrease to near control levels by 4 h. Expression levels of six stress marker genes in general for all groups showed down regulation over a 9-day sampling period. In contrast, expression levels for immune response genes in the head kidney showed significant up-regulation for all eight genes tested for both vaccinated groups whereas the non-vaccinated group showed up-regulation for only MHC-II and IL-6b in comparison to the control. Both the vaccination procedure and the administration of the vaccine itself were factors mediating changes in gene expression consistent with fish being susceptible to natural occurring saprolegniosis following vaccination., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Kelps are key primary producers of cold and temperate marine coastal ecosystems and exhibit systemic defences against pathogens. Yet, the cellular mechanisms underpinning their immunity remain to be elucidated. We investigated the time course of infection of the kelp Macrocystis pyrifera by the oomycete Anisolpidium ectocarpii using TEM, in vivo autophagy markers and autophagy inhibitors. Over several infection cycles, A. ectocarpii undergoes sequential physiological shifts sensitive to autophagy inhibitors. Initially lipid-rich, pathogen thalli become increasingly lipid-depleted; they subsequently tend to become entirely abortive, irrespective of their lipid content. Moreover, infected algal cells mount local defences and can directly eliminate the pathogen by xenophagy. Finally, autophagy-dependent plastid recycling is induced in uninfected host cells. We demonstrate the existence of local, inducible autophagic processes both in the pathogen and infected host cells, which result in the restriction of pathogen propagation. We also show the existence of a systemic algal response mediated by autophagy. We propose a working model accounting for all our observations, whereby the outcome of the algal-pathogen interaction (i.e. completion or not of the pathogen life cycle) is dictated by the induction, and possibly the mutual hijacking, of the host and pathogen autophagy machineries., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Macrocystis pyrifera and Lessonia spicata are economically and ecologically relevant brown seaweeds that recently have been classified as members of two separated families within Laminariales (kelps). Here we describe for the first time the Macrocystis pyrifera x Lessonia spicata hybridization in the wild (Chiloe Island, Southeastern Pacific), where populations of the two parents exist sympatrically. Externally, this hybrid exhibited typical features of its parents M. pyrifera (cylindrical and flexible distal stipes, serrate frond margins and presence of sporophylls) and L. spicata (rigid and flat main stipe and first bifurcation), as well as intermediate features between them (thick unfused haptera in the holdfast). Histological sections revealed the prevalence of mucilage ducts within stipes and fronds (absent in Lessonia) and fully developed unilocular sporangia in the sporophylls. Molecular analyses confirmed the presence of the two parental genotypes for ITS1 nrDNA and the M. pyrifera genotype for two predominantly maternally inherited cytoplasmic markers (COI and rbcLS spacer) in the tissue of the hybrid. A metabolome-wide approach revealed that this hybrid is more chemically reminiscent to M. pyrifera. Nevertheless, several hits were identified as Lessonia exclusive or more remarkably, not present in any of the parent. Meiospores developed into apparently fertile gametophytes, which gave rise to F1 sporophytes that reached several millimeters before suddenly dying. In-vitro reciprocal crossing of Mar Brava gametophytes from both species revealed that although it is rare, interfamilial hybridization between the two species is possible but mostly overcome by pseudogamy of female gametophytes.

Aquatic zoosporic diseases are threatening global biodiversity and ecosystem services, as well as economic activities. Current means of controlling zoosporic diseases are restricted primarily to chemical treatments, which are usually harmful or likely to be ineffective in the long term. Furthermore, some of these chemicals have been banned due to adverse effects. As a result, there is a need for alternative methods with minimal side-effects on the ecosystem or environment. Here, we integrate existing knowledge of three poorly interconnected areas of disease research - amphibian conservation, aquaculture, and plankton ecology - and arrange it into seven biological concepts to control zoosporic diseases. These strategies may be less harmful and more sustainable than chemical approaches. However, more research is needed before safe application is possible., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Oomycetes are eukaryotic pathogens infecting animals and plants. Amongst them Saprolegnia parasitica is a fish pathogenic oomycete causing devastating losses in the aquaculture industry. To secure fish supply, new drugs are in high demand and since fish experiments are time consuming, expensive and involve animal welfare issues the search for adequate model systems is essential. Galleria mellonella serves as a heterologous host model for bacterial and fungal infections. This study extends the use of G. mellonella for studying infections with oomycetes. Saprolegniales are highly pathogenic to the insects while in contrast, the plant pathogen Phytophthora infestans showed no pathogenicity. Melanisation of hyphae below the cuticle allowed direct macroscopic monitoring of disease progression. However, the melanin response is not systemic as for other pathogens but instead is very local. The mortality of the larvae is dose-dependent and can be induced by cysts or regenerating protoplasts as an alternative source of inoculation., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Phytomyxea are obligate endoparasites of angiosperm plants and Stramenopiles characterised by a complex life cycle. Here Maullinia braseltonii sp. nov., an obligate parasite infecting the bull kelp Durvillaea (Phaeophyceae, Fucales) from the South-Eastern Pacific (Central Chile and Chiloe Island) and South-Western Atlantic (Falkland Islands, UK) is described. M. braseltonii causes distinct hypertrophies (galls) on the host thalli making it easily identifiable in the field. Sequence comparisons based on the partial 18S and the partial 18S-5.8S-28S regions confirmed its placement within the order Phagomyxida (Phytomyxea, Rhizaria), as a sister species of the marine parasite Maullinia ectocarpii, which is also a parasite of brown algae. The development of resting spores in M. braseltonii is described by light and electron microscopy and confirmed by FISH experiments, which visually showed the differential expression of the 28S non-coding gene, strongly in early plasmodia and weakly in late cysts. M. braseltonii is, so far, the only phytomyxean parasite of brown algae for which the formation of resting spores has been reported, and which is widely distributed in Durvillaea stocks from the Southeastern Pacific and Southwestern Atlantic., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

When plant-pathogenic oomycetes infect their hosts, they employ a large arsenal of effector proteins to establish a successful infection. Some effector proteins are secreted and are destined to be translocated and function inside host cells. The largest group of translocated proteins from oomycetes is the RxLR effectors, defined by their conserved N-terminal Arg-Xaa-Leu-Arg (RxLR) motif. However, the precise role of this motif in the host cell translocation process is unclear. Here, detailed biochemical studies of the RxLR effector AVR3a from the potato pathogen Phytophthora infestans are presented. Mass spectrometric analysis revealed that the RxLR sequence of native AVR3a is cleaved off prior to secretion by the pathogen and the N terminus of the mature effector was found likely to be acetylated. High-resolution NMR structure analysis of AVR3a indicates that the RxLR motif is well accessible to potential processing enzymes. Processing and modification of AVR3a is to some extent similar to events occurring with the export element (PEXEL) found in malaria effector proteins from Plasmodium falciparum These findings imply a role for the RxLR motif in the secretion of AVR3a by the pathogen, rather than a direct role in the host cell entry process itself., (© 2017 American Society of Plant Biologists. All rights reserved.)

The three-spined stickleback (Gasterosteus aculeatus) is a model organism with an extremely well-characterized ecology, evolutionary history, behavioural repertoire and parasitology that is coupled with published genomic data. These small temperate zone fish therefore provide an ideal experimental system to study common diseases of coldwater fish, including those of aquacultural importance. However, detailed information on the culture of stickleback parasites, the establishment and maintenance of infections and the quantification of host responses is scattered between primary and grey literature resources, some of which is not readily accessible. Our aim is to lay out a framework of techniques based on our experience to inform new and established laboratories about culture techniques and recent advances in the field. Here, essential knowledge on the biology, capture and laboratory maintenance of sticklebacks, and their commonly studied parasites is drawn together, highlighting recent advances in our understanding of the associated immune responses. In compiling this guide on the maintenance of sticklebacks and a range of common, taxonomically diverse parasites in the laboratory, we aim to engage a broader interdisciplinary community to consider this highly tractable model when addressing pressing questions in evolution, infection and aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Oomycetes, or water moulds, are fungal-like organisms phylogenetically related to algae. They cause devastating diseases in both plants and animals. Here, we describe seven oomycete species that are emerging or re-emerging threats to agriculture, horticulture, aquaculture and natural ecosystems. They include the plant pathogens Phytophthora infestans, Phytophthora palmivora, Phytophthora ramorum, Plasmopara obducens, and the animal pathogens Aphanomyces invadans, Saprolegnia parasitica and Halioticida noduliformans For each species, we describe its pathology, importance and impact, discuss why it is an emerging threat and briefly review current research activities.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'., (© 2016 The Author(s).)

Global climate change is expected to alter the polar bioregions faster than any other marine environment. This study assesses the biodiversity of seaweeds and associated eukaryotic pathogens of an established study site in northern Baffin Island (72° N), providing a baseline inventory for future work assessing impacts of the currently ongoing changes in the Arctic marine environment. A total of 33 Phaeophyceae, 24 Rhodophyceae, 2 Chlorophyceae, 12 Ulvophyceae, 1 Trebouxiophyceae, and 1 Dinophyceae are reported, based on collections of an expedition to the area in 2009, complemented by unpublished records of Robert T. Wilce and the first-ever photographic documentation of the phytobenthos of the American Arctic. Molecular barcoding of isolates raised from incubated substratum samples revealed the presence of 20 species of brown seaweeds, including gametophytes of kelp and of a previously unsequenced Desmarestia closely related to D. viridis, two species of Pylaiella, the kelp endophyte Laminariocolax aecidioides and 11 previously unsequenced species of the Ectocarpales, highlighting the necessity to include molecular techniques for fully unraveling cryptic algal diversity. This study also includes the first records of Eurychasma dicksonii, a eukaryotic pathogen affecting seaweeds, from the American Arctic. Overall, this study provides both the most accurate inventory of seaweed diversity of the northern Baffin Island region to date and can be used as an important basis to understand diversity changes with climate change., (© 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.)

Pathogens are increasingly being recognized as key evolutionary and ecological drivers in marine ecosystems. Defence mechanisms of seaweeds, however, have mostly been investigated by mimicking infection using elicitors. We have established an experimental pathosystem between the genome brown model seaweed Ectocarpus siliculosus and the oomycete Eurychasma dicksonii as a powerful new tool to investigate algal responses to infection. Using proteomics, we identified 21 algal proteins differentially accumulated in response to Eu. dicksonii infection. These include classical algal stress response proteins such as a manganese superoxide dismutase, heat shock proteins 70 and a vanadium bromoperoxidase. Transcriptional profiling by qPCR confirmed the induction of the latter during infection. The accumulation of hydrogen peroxide was observed at different infection stages via histochemical staining. Inhibitor studies confirmed that the main source of hydrogen peroxide is superoxide converted by superoxide dismutase. Our data give an unprecedented global overview of brown algal responses to pathogen infection, and highlight the importance of oxidative stress and halogen metabolism in these interactions. This suggests overlapping defence pathways with herbivores and abiotic stresses. We also identify previously unreported actors, in particular a Rad23 and a plastid-lipid-associated protein, providing novel insights into the infection and defence processes in brown algae., (© 2015 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

A new geographic record of the oomycete Olpidiopsis feldmanni infecting the tetrasporophytic stage of the red alga Asparagopsis sp. from the Adriatic Sea, confirmed through morphological identification, allowed us to expand previous observations of this organism. Ultrastructural investigations of environmental material showed a large central vacuole and a cell wall thicker than previously reported from other basal oomycete pathogens of algae. Phylogenetic analysis closely associates O. feldmanni to O. bostrychiae concurrent with structural observations. This constitutes the first genetic characterisation of an Olpidiopsis species that was initially described before 1960, adding to the genetic data of 3 other marine Olpidiopsis species established and genetically characterised in the last 2 decades. The paper discusses concurrences of the ultrastructural observations made here and in previous studies of the marine Olpidiopsis species with those made on the freshwater species.

In this study, we assessed the development (formation, taxis and settlement) of eukaryotic zoospores under different regimes of exposure to polycyclic aromatic hydrocarbons (PAHs), which imitated environmental scenarios of pollution and bioremediation. With this aim, we used an oomycete, Pythium aphanidermatum, as a source of zoospores and two PAH-degrading bacteria (Mycobacterium gilvum VM552 and Pseudomonas putida G7). The oomycete and both bacteria were not antagonistic, and zoospore formation was diminished only in the presence of the highest bacterial cell density (10(8)-10(10) colony-forming units mL(-1)). A negative influence of PAHs on zoospore formation and taxis was observed when PAHs were exposed in combination with organic solutions and polar solvents. Co-exposure of PAHs with non-polar solvents [hexadecane (HD) and 2,2,4,4,6,8,8-heptamethylnonane (HMN)] did not affect zoospore settlement at the interfaces of the organic solvents and water. However, zoospores settled and created mycelial networks only at HD-water interfaces. Both bacteria diminished the toxic influence of PAHs on zoospore formation and taxis, and they did not interrupt zoospore settlement. The results suggest that zoospore development could be applicable for toxicity assessment of PAHs and enhancement of their bioavailability. Microbial interactions during both swimming modes and community formation at pollutant interfaces were revealed as major factors that have potential relevance to bioremediation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1β1 [IL-1β1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin [corrected] E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments., (Copyright © 2014. Published by Elsevier Ltd.)

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments., (Copyright © 2013. Published by Elsevier Ltd.)

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrumsmall tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation., (Copyright © 2013 The British Mycological Society. All rights reserved.)

Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The mechanism of translocation of RxLR effectors from plant pathogenic oomycetes into the cytoplasm of their host is currently the object of intense research activity and debate. Here, we report the biochemical and thermodynamic characterization of the Phytophthora infestans effector AVR3a in vitro. We show that the amino acids surrounding the RxLR leader mediate homodimerization of the protein. Dimerization was considerably attenuated by a localized mutation within the RxLR motif that was previously described to prevent translocation of the protein into host. Importantly, we confirm that the reported phospholipid-binding properties of AVR3a are mediated by its C-terminal effector domain, not its RxLR leader. However, we show that the observed phospholipid interaction is attributable to a weak association with denatured protein molecules and is therefore most likely physiologically irrelevant.

The oomycete Saprolegnia parasitica causes significant losses in the aquaculture industry, mainly affecting salmon, trout and catfish. Since the ban of malachite green, effective control measures are currently not available prompting a re-evaluation of the potential for immunological intervention. In this study, the immune response of salmonid cells is investigated at the transcript level, by analysis of a large set of immune response genes in four different rainbow trout cell lines (RTG-2, RTGill, RTL and RTS11) upon infection with S. parasitica. Proinflammatory cytokine transcripts were induced in all four cell lines, including IL-1β1, IL-8, IL-11, TNF-α2, as well as other components of the innate defences, including COX-2, the acute phase protein serum amyloid A and C-type lectin CD209a and CD209b. However, differences between the four cell lines were found. For example, the fold change of induction was much higher in the epithelial RTL and macrophage-like RTS11 cell lines compared to the fibroblast cell lines RTG-2 and RTGill. Several antimicrobial peptides (AMPs) were also up-regulated in response to Saprolegnia infection, including hepcidin and cathelicidin 1 (rtCATH1) and 2 (rtCATH2). An rtCATH2 peptide was synthesised and tested for activity and whilst it showed no killing activity for zoospores, it was able to delay sporulation of S. parasitica. These results demonstrate that particular immune genes are up-regulated in response to S. parasitica infection and that AMPs may play a crucial role in the first line of defence against oomycetes in fish., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

Background: Pythium 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 of these 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.

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.

SUMMARY Gene silencing, triggered by double-stranded RNA (dsRNA), has proved to be a valuable tool for determining and confirming the function of genes in many organisms. For Phytophthora infestans, the cause of late blight on potato and tomato, gene silencing strategies have relied on stable transformation followed by spontaneous silencing of both the endogenous gene and the transgene. Here we describe the first application of transient gene silencing in P. infestans, by delivering in vitro synthesized dsRNA into protoplasts to trigger silencing. A marker gene, gfp, and two P. infestans genes, inf1 and cdc14, both of which have been silenced previously, were selected to test this strategy. Green fluorescent protein (GFP) fluorescence was reduced in regenerating protoplasts up to 4 days after exposure to gfp dsRNA. A secondary reduction in expression of all genes tested was not fully activated until 12-17 days after introduction of the respective homologous dsRNAs. At this time after exposure to dsRNA, reduced GFP fluorescence in gfp dsRNA-treated lines, and reduced INF1 production in inf1 dsRNA-treated lines, was observed. Introduction of dsRNA for the stage-specific gene, cdc14, yielded the expected phenotype of reduced numbers of sporangia when cdc14 expression was significantly reduced compared with control lines. Silencing was shown to be sequence-specific, because analysis of inf1 expression in gfp-silenced lines yielded wild-type levels of gene expression. This report shows that transient gene silencing can be used to generate detectable phenotypes in P. infestans and should provide a high-throughput tool for P. infestans functional genomics.