Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

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.     

Galleria melonella as an experimental in vivo host model for the fish-pathogenic oomycete Saprolegnia parasitica

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.     

Moving targets: rapid evolution of oomycete effectors

Plant pathogenic microbes secrete proteins known as effectors, which enter the cytoplasm of plant cells and suppress host defences. Known effectors in oomycete pathogens possess an RXLR-EER motif in their amino acid sequence that is necessary for transport of the effector into a host plant cell. A large number of putative effectors have now been identified in oomycete genomes, the sequences of which show evidence of diversifying selection at their C terminus. Here, we describe recent progress in characterizing RXLR-EER effectors and discuss why so many of these rapidly evolving proteins are encoded by the genomes of plant pathogenic oomycetes.     

A domain-centric analysis of oomycete plant pathogen genomes reveals unique protein organization

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.     

The molecular dialog between oomycete effectors and their plant and animal hosts

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.     

Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.     

The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

Reprint of: The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

Sterol biosynthesis in oomycete pathogens

Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores, but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such as diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Saprolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs, suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales.Key words: azoles, fungicides, root rot, elicitin, Saprolegnia, chromoalveolates     

Structural and phylogenetic analyses of the GP42 transglutaminase from Phytophthora sojae reveal an evolutionary relationship between oomycetes and marine Vibrio bacteria

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca²⁺-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.     

Molecular Assays for Detecting Aphanomyces invadans in Ulcerative Mycotic Fish Lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.     

Deciphering microbial landscapes of fish eggs to mitigate emerging diseases

Animals and plants are increasingly suffering from diseases caused by fungi and oomycetes. These emerging pathogens are now recognized as a global threat to biodiversity and food security. Among oomycetes, Saprolegnia species cause significant declines in fish and amphibian populations. Fish eggs have an immature adaptive immune system and depend on nonspecific innate defences to ward off pathogens. Here, meta-taxonomic analyses revealed that Atlantic salmon eggs are home to diverse fungal, oomycete and bacterial communities. Although virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegniato salmon eggs. These results highlight that fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate emerging diseases.     

A New Marine Species of Miracula (Oomycota) Parasitic to Minidiscus sp. in Iceland†

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.     

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.     

Common processes in pathogenesis by fungal and oomycete plant pathogens,described with Gene Ontology terms

Plant diseases caused by fungi and oomycetes result in significant economic losses every year. Although phylogenetically distant, the infection processes by these organisms share many common features. These include dispersal of an infectious particle, host adhesion, recognition, penetration, invasive growth, and lesion development. Previously, many of these common processes did not have corresponding Gene Ontology (GO) terms. For example, no GO terms existed to describe processes related to the appressorium, an important structure for infection by many fungi and oomycetes. In this mini-review, we identify common features of the pathogenic processes of fungi and oomycetes and create a pathogenesis model using 256 newly developed and 38 extant GO terms, with an emphasis on the appressorium and signal transduction. This set of standardized GO terms provides a solid base to further compare and contrast the molecular underpinnings of fungal and oomycete pathogenesis.