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Aphanomyces invadans is a eukaryotic pathogen and the causative agent of Epizootic Ulcerative Syndrome (EUS) in fish and is responsible for mortalities of up to 100% in aquaculture. A. invadans was first discovered in Japan in 1971, and since then it has been found in Australia, North America, Southern African countries and Asia. Methods for the correct identification of A. invadans are well established now and involve PCR-based detection and microscopy. However, the pathogenesis of A. invadans is poorly understood. Environmental stress (mainly temperature) and the associated immunocompromised fish seem to induce infections of A. invadans and outbreaks of EUS. Understanding the process of infection in more depth is fundamental for the discovery of novel effective treatments to combat the disease. In this review, we discuss morphological characteristics of A. invadans and its pathogenicity as well as various approaches of treatment. [ABSTRACT FROM AUTHOR]

Oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that include some of the most notorious pathogens of plants and animals. Through the deployment of a remarkably diverse array of effector proteins, oomycete pathogens succeed to overcome host defences and cause infection. Effectors can operate extracellularly or enter living cells where they target diverse subcellular compartments. Genome sequence information indicates that oomycetes express several hundred host-translocating effectors potentially targeting a myriad of host processes. To counteract, plants rely on a wide variety of extra- and intracellular immune receptors facilitating pattern-triggered and effector-triggered immunity, respectively. Similarly, effectors from animal pathogenic oomycetes also target host immune response pathways, which in turn causes the activation of the humoral and adaptive immune system. In this review, we compare plant and animal pathogenic oomycete effectors regarding their type, function, genetic diversity, as well as host responses. [ABSTRACT FROM AUTHOR]

Cadherins, a group of molecules typically associated with planar cell polarity and Wnt signalling, have been little reported outside of the animal kingdom. Here, we identify a new family of cadherins in the Stramenopiles, termed Nonagonal after their 9 transmembrane passes, which contrast to the one or seven passes found in other known cadherin families. Manual curation and experimental validation reveal two subclasses of nonagonal cadherins, depending on the number of uninterrupted extracellular cadherin (EC) modules presented. Firstly, shorter mono-exonic, unimodular, protein models, with 3 to 12 EC domains occur as duplicate paralogs in the saprotrophic Labyrinthulomycetes Aurantiochytrium limanicum and Schizochytrium aggregatum , the gastrointestinal Blastocystis hominis (Blastocystae) and as a single copy gene in the autotrophic Pelagophyte Aureococcus anophagefferens . Larger, single copy, multi-exonal, tri-modular protein models, with up to 72 EC domain in total, are found in the Oomycete genera Albugo , Phytophthora, Pythium and Eurychasma . No homolog was found in the closely related autotrophic Phaeophyceae (brown algae) or Bacillariophyceae (diatoms), nor in several genera of plant and animal pathogenic oomycetes ( Aphanomyces , Saprolegnia and Hyaloperonospora ). This potential absence was further investigated by synteny analysis of the genome regions flanking the cadherin gene models, which are found to be highly variable. Novel to this new cadherin family is the presence of intercalated laminin and putative carbohydrate binding in tri-modular oomycete cadherins and at the N-terminus of thraustochytrid proteins. As we were unable to detect any homologs of proteins involved in signalling pathways where other cadherin families are involved, we present a conceptual hypothesis on the function of nonagonal cadherin based around the presence of putative carbohydrate binding domains. [ABSTRACT FROM AUTHOR]

Abstract: A very large number of diseases in agriculture and aquaculture are caused by fungal-like micro-organisms, classified as Oomycetes or water moulds. Collectively, oomycetes represent a huge threat to global food security. In addition, several oomycetes can cause environmental disasters, by wiping out native species of trees, crustacians and amphibians. The group representing the aquatic oomycetes are relatively understudied in contrast to their terrestrial counterparts that predominantly infect plants (e.g. Phytophthora, Pythium and Bremia spp.). This review focuses on the unique characteristics of two aquatic Oomycetes, Saprolegnia parasitica and Saprolegnia diclina with respect to their impact on aquaculture, animal health and the surrounding environment. The species characteristics, ecology, biology, infectivity and identification methods are described and the latest research insights are discussed. [Copyright &y& Elsevier]

Abstract: Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis. [Copyright &y& Elsevier]

Abstract: The most notorious oomycetes, such as Phytophthora infestans, are pathogens of higher plants, although numerous other species of these fungal-like microorganisms infect algae, crustacea, nematodes, fish and mammals. While there is now ample evidence that oomycetes and fungi deliver effector proteins inside the host cell during infection, like bacteria using the well characterised type III secretion system, the mechanism employed by eukaryotic pathogens remains unclear. In oomycetes this process depends on an N-terminal motif defined by a short conserved amino acid sequence (RxLR) located near the signal peptide of many secreted proteins. This motif resembles the host-cell targeting signal found in virulence proteins from the malaria parasite Plasmodium falciparum (RxLxE/D/Q). This review will focus on the recent findings contributing to the understanding of the delivery of oomycete effector molecules into the host cells, with emphasis on how they compare with various models proposed for filamentous fungi and the malaria parasite. [ABSTRACT FROM AUTHOR]

Abstract: Oomycetes cause destructive diseases on both animals and plants. The epidemic spread of oomycete diseases is primarily based on rapid dispersal from host to host by free swimming zoospores. These single-nucleated spores are formed in sporangia and are only released in aqueous environments. Oomycetes are classified in the Kingdom of the Stramenopiles or Chromista, which is comprised of several organisms, including the golden brown algae. The unique shared attribute found in most Stramenopiles is the morphology of the zoospores and especially the structure of their two flagella. They have one tinsel flagellum, and one whiplash flagellum. Only the tinsel flagellum has distinctive flagellar hairs. Zoospore formation can occur within minutes and it is considered one of the fastest developmental processes in any biological system. Once released from the sporangium they are able to exhibit chemotactic responses, electrotaxis, and autotaxis or autoaggregation to target new hosts for infection. Here we discuss the latest discoveries in the development and biology of the oomycete zoospore. [Copyright &y& Elsevier]

Abstract: Water moulds (oomycetes) of the order Saprolegniales, such as Saprolegnia and Aphanomyces species, are responsible for devastating infections on fish in aquaculture, fish farms and hobby fish tanks. Members of the genus Saprolegnia cause Saprolegniosis, a disease that is characterised by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green, an organic dye that is very efficient at killing the pathogen. However, the use of malachite green has been banned worldwide due to its carcinogenic and toxicological effects and this has resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new alternative control strategies. [Copyright &y& Elsevier]

This review discusses recent advances in research into plant pathogenic oomycetes with an emphasis on root-infecting species. We focus on aspects of host targeting, mycoparasitism, and the development of molecular techniques that enable functional dissection of key genes of this economically important group of pathogens, including genomics, proteomics and gene silencing. We have not incorporated aspects relating to host resistance, research carried out into downy mildews, and other phyloplane oomycetes, unless there is also a specific relevance to the root-oomycete research community. The analysis of the asexual life stages of these organisms from zoosporogenesis through zoospore liberation, host targeting, encystment, germination, and the formation of appressoria-like structures in the rhizosphere offer significant potential in the establishment of new approaches for the treatment of disease in these organisms. The advent of appropriate molecular tools is now enabling the molecular analysis of these developmental stages to begin in earnest and will stimulate research into an economically important but scientifically neglected group of organisms. [Copyright &y& Elsevier]

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. • Description of an RNAi-based silencing protocol for A. invadans. • Galleria mellonella as an infection model for A. invadans. • Virulence factors of Aphanomyces invadans are partially dependent on conventional protein secretion. [ABSTRACT FROM AUTHOR]

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. Aquatic zoosporic diseases pose a threat to biodiversity, ecosystem services, and economic activities. Current control methods are based on the use of chemicals that have large negative side-effects. To support the development of alternative biological control strategies, we outline seven general biological concepts to control zoosporic diseases. We discuss some of the research required to successfully apply biological control strategies in the future. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

Abstract: 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 oligandrum small 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: Proteins embedded in the cell wall and plasma membrane of filamentous oomycetes and fungi provide a means by which these organisms can interact with their local environment. However, cell wall and membrane proteins have often proved difficult to isolate using conventional proteomic techniques. Here we have used liquid chromatography tandem mass spectrometry (LC-MS/MS) to facilitate rapid and sensitive quantification of the cell wall proteome. We report the use of LC-MS/MS to identify differentially regulated proteins from the cell walls of three different lifecycle stages of the oomycete plant pathogen Phytophthora infestans: non-sporulating vegetative mycelium, sporulating mycelium, and germinating cysts with appressoria. We have also used quantitative real-time RT-PCR to confirm that the transcripts corresponding to some of these proteins, namely those identified in cell walls of germinating cysts with appressoria, accumulate differentially throughout the lifecycle. These proteins may, therefore, be important for pre-infective development and early pathogenicity. Up to 31 covalently and non-covalently bound cell wall-associated proteins were identified. All of the proteins identified in germinating cysts with appressoria, and several of those from mycelial fractions, were classified as putative effector or pathogen-associated molecular pattern (PAMP) molecules, including members of the CBEL family, the elicitin family, the crinkler (CRN) family and two transglutaminases. Thus, the cell wall of P. infestans may represent an important reservoir for surface-presented, apoplastic effectors or defence activation molecules. Proteins predicted to be cell surface proteins included IPI-B like proteins, mucins, cell wall-associated enzymes and annexin family members. Additionally we identified up to 27 membrane-associated proteins from Triton X-114 phase partitioned mycelial membrane preparations, producing the first inventory of oomycete membrane-associated proteins. Four of these proteins are small Rab-type G-proteins and several are associated with secretion. [Copyright &y& Elsevier]

Abstract: Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control. [Copyright &y& Elsevier]