Search

Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.

Oomycetes cause devastating diseases on plants and animals. They cause major yield losses in many crop plants and their control heavily depends on agrochemicals. This is certainly true for the potato late blight pathogen Phytophthora infestans. Strong concerns about adverse effects of agrochemicals on food safety and environment are incentives for the development of novel, environmental friendly control strategies preferably based on natural products. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes including Phytophthora. CLPs lyse zoospores, inhibit mycelial growth and effectively reduce late blight disease. In order to unravel how Phytophthora senses CLPs and other environmental signals we follow two approaches. On the one hand, we monitor genome wide changes in gene expression induced by CLPs with the aim to identify the cellular pathways targeted by CLPs. On the other hand, we analyse components of ubiquitous signal transduction pathways with the aim to identify features that are unique for Phytophthora or oomycetes and, hence, could be suitable targets for novel anti-oomycete agents. Mining and comparing whole genome sequences have revealed that Phytophthora harbours many novel phospholipid modifying enzymes, unique for oomycetes. They have aberrant combinations of catalytic and regulatory domains occasionally combined with transmembrane domains. The latter resemble receptors that might be activated by extracellular ligands. Phospholipids, the substrates of these enzymes, are structural membrane components that also function in signalling. Together these findings open new avenues of research aimed at target-discovery in oomycetes.

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.

In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P. sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.

In eukaryotes phospholipase D (PLD) is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately 1/10 being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

A correct interpretation of isozyme patterns can be seriously hampered by the lack of supporting genetic data. The availability of the complete genome sequence of Phytophthora ramorum, enabled us to correlate isozyme profiles with the gene models predicted for these enzymes. Thirty-nine P. ramorum strains were characterised employing isozyme analysis on malate dehydrogenase (MDH), NADP-dependent malic enzyme (MDHP), 6-phosphogluconate dehydrogenase (PGD), glucosephosphate isomerase (GPI) and lactate dehydrogenase (LDH) comprising nine putative loci. One isozyme band was enzymatically stained for PGD whereas multiple bands were detected for GPI, MDH, MDHP and LDH. All putative loci were monomorphic except for Ldh-2. Genome mining revealed that the assembled genome sequences of P. ramorum and P. sojae each contain one Gpi and one Pgd gene model. For MDH, two gene models were identified, encoding a cytosolic and mitochondrial type, respectively. Also for MDHP P. ramorum has two gene models that are both duplicated in P. sojae. Both species contain six Ldh gene models, including pseudogenes. The Ldh gene models are clustered and located in regions that show a high level of conserved synteny. This study demonstrates that insight into the gene models encoding isozymes helps to interpret isozyme profiles in Phytophthora.

Phospholipids are cellular membrane components in eukaryotic cells that execute many important roles in signaling. Genes encoding enzymes required for phospholipid signaling and metabolism have been characterized in several organisms, but only a few have been described for oomycetes. In this study, the genome sequences of Phytophthora sojae and P. ramorum were explored to construct a comprehensive genomewide inventory of genes involved in the most universal phospholipid signaling pathways. Several genes and gene families were annotated, including those encoding phosphatidylinositol synthase (PIS), phosphatidy-linositol (phosphate) kinase (PI[P]K), diacylglycerol kinase (DAG), and phospholipase D (PLD). The most obvious missing link is a gene encoding phospholipase C (PLC). In all eukaryotic genomes sequenced to date, PLC genes are annotated based on certain conserved features; however, these genes seem to be absent in Phytophthora spp. Analysis of the structural and regulatory domains and domain organization of the predicted isoforms of PIS, PIK, PIPK, DAG, and PLD revealed many novel features compared with characterized representatives in other eukaryotes. Examples are transmembrane proteins with a C-terminal catalytic PLD domain, secreted PLD-like proteins, and PIPKs that have an N-terminal G-protein-coupled receptor-transmembrane signature. Compared with other sequenced eukaryotes, the genus Phytophthora clearly has several exceptional features in its phospholipid-modifying enzymes.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum . Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosis-inducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

G-protein coupled receptors (GPCR) and phosphatidylinositol phosphate kinases (PIPK) are important key switches in signal transduction pathways. A novel class of proteins was identified in the genomes of two unrelated organisms that harbor both a GPCR and a PIPK domain. Dictyostelium discoideum contains one GPCR¿PIPK, which is crucial in cell-density sensing, and the genomes of Phytophthora sojae and Phytophthora ramorum each encode twelve GPCR¿PIPKs. Intriguingly, these are currently the only species that have these two domains combined in one protein. Here, the structural and regulatory characteristics of GPCR¿PIPKs are presented and discussed. It is hypothesized that, upon activation, GPCR¿PIPKs are able to trigger heterotrimeric G-protein signaling and phosphoinositide second-messenger synthesis.

Phospholipase D (PLD) is a ubiquitous enzyme in eukaryotes that participates in various cellular processes. Its catalytic domain is characterized by two HKD motifs in the C-terminal part. Until now, two subfamilies were recognized based on their N-terminal domain structure. The first has a PX domain in combination with a PH domain and is designated as PXPH-PLD. Members of the second subfamily, named C2-PLD, have a C2 domain and have, so far, only been found in plants. Here we describe a novel PLD subfamily that we identified in Phytophthora, a genus belonging to the class oomycetes and comprising many important plant pathogens. We cloned Pipld1 from Phytophthora infestans and retrieved full-length sequences of its homologues from Phytophthora sojae and Phytophthora ramorum genome databases. Their promoters contain two putative regulatory elements, one of which is highly conserved in all three genes. The three Phytophthora pld1 genes encode nearly identical proteins of around 1807 amino acids, with the two characteristic HKD motifs in the C-terminal part. Homology of the predicted proteins with known PLDs however is restricted to the two catalytic HKD motifs and adjacent domains. In the N-terminal part Phytophthora PLD1 has a PX-like domain, but it lacks a PH domain. Instead the N-terminal region contains five putative membrane spanning domains suggesting that Phytophthora PLD1 is a transmembrane protein. Since Phytophthora PLD1 cannot be categorized in one of the two existing subfamilies we propose to create a novel subfamily named PXTM-PLD.

SUMMARY In many plant pathogens heterotrimeric G-proteins are essential signalling components involved in development and pathogenicity. In the late blight oomycete pathogen Phytophthora infestans the G-protein alpha subunit PiGPA1 controls zoospore motility and is required for virulence. To identify G-protein targets and signalling pathways downstream of PiGPA1, we used an optimized cDNA-AFLP protocol for analysing gene expression profiles in hypovirulent P. infestans strains that were previously generated by silencing the Pigpa1 gene. First, expression profiles in sporangia and mycelium of the wild-type strain were compared, and this revealed a substantial number of mycelium- or sporangia-specific transcript derived fragments (TDFs). Subsequently, profiles in sporangia of wild-type, Pigpa1-silenced mutants and of a strain expressing a constitutively active form of PiGPA1 were compared. From a total of 2860 TDFs, 92 were down- and 19 up-regulated in the Pigpa1-silenced mutants. A subset of the differential TDFs was cloned and sequenced, and homology searches were carried out against Phytophthora EST and genomic databases and the NCBI database. cDNA-AFLP expression profiles were verified by Northern blot analysis or RT-PCR. The power of cDNA-AFLP for the identification of target genes in knock-down or gain-of-function mutants is discussed.

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., Author Summary Fish are an increasingly important source of animal protein globally, with aquaculture production rising dramatically over the past decade. Saprolegnia is a fungal-like oomycete and one of the most destructive fish pathogens, causing millions of dollars in losses to the aquaculture industry annually. Saprolegnia has also been linked to a worldwide decline in wild fish and amphibian populations. Here we describe the genome sequence of the first animal pathogenic oomycete and compare the genome content with the available plant pathogenic oomycetes. We found that Saprolegnia lacks the large effector families that are hallmarks of plant pathogenic oomycetes, showing evolutionary adaptation to the host. Moreover, Saprolegnia harbors pathogenesis-related genes that were derived by lateral gene transfer from the host and other animal pathogens. The retrotransposon LINE family also appears to be acquired from animal lineages. By transcriptome analysis we show a high rate of allelic variation, which reveals rapidly evolving genes and potentially adaptive evolutionary mechanisms coupled to selective pressures exerted by the animal host. The genome and transcriptome data, as well as subsequent biochemical analyses, provided us with insight in the disease process of Saprolegnia at a molecular and cellular level, providing us with targets for sustainable control of Saprolegnia.

Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs) are produced in a wide variety of species (from bacteria to humans) and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively) were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the Aβ peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during their translocation into the host red blood cell. Since the translocation of Phytophthora effector proteins is currently a topic of intense research activity, the identification in Phytophthora of potential functional homologues of plasmepsin V would appear worthy of investigation. Indeed, elucidation of the physiological roles of the APs identified here offers areas for future study. The significant revision of gene models and detailed annotation presented here should significantly facilitate experimental design.