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Fusarium wilt of banana (FWB) is a serious soil-borne fungal disease. In the previous century, FWB already destroyed Gros Michel-based banana cultures in Central America, and currently, the disease threatens all major banana-producing regions of the world. The causal agents of these epidemics, however, are diverse. Gros Michel was infected by a wide range of Fusarium species, the so-called Race 1 strains, whereas the contemporary Cavendish-based cultures are affected by Fusarium odoratissimum, colloquially called Tropical Race 4 (TR4). TR4 was reported in Mozambique on two commercial banana farms in 2013, but no incursions were found outside the farm boundaries in 2015, suggesting that the disease was under control. Here we report the presence of TR4 outside of these farm boundaries. We obtained fungal samples from 13 banana plants in smallholder and roadside plantings at various locations throughout northern Mozambique. These samples tested positive for TR4 by molecular diagnostics and in greenhouse pathogenicity assays. The results were confirmed with reisolations, thereby completing Koch’s postulates. To study the diversity of TR4 isolates in Mozambique, we selected five samples for whole-genome sequencing. Comparison with a global collection of TR4 samples revealed very little genetic variation, indicating that the fungus is clonally spreading in Mozambique. Furthermore, isolates from Mozambique are clearly genetically separated from other geographic incursions, and thus we cannot trace the origin of TR4 in Mozambique. Nevertheless, our data demonstrates the dissemination of TR4 in Mozambique, underscoring the failure of disease management strategies. This threatens African banana production.

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.

Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo47 and Fo618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations.

The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.

Sensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling. Heterotrimeric G-protein subunits activated by G-protein-coupled receptors (GPCRs) are typical stimulators of phospholipid signaling enzymes such as phosphatidylinositol phosphate kinases (PIPKs) or phospholipase C (PLC). However, a direct connection between the two pathways likely exists in oomycetes and slime molds, as they possess a unique class of GPCRs that have a PIPK as an accessory domain. In principle, these so-called GPCR-PIPKs have the capacity of perceiving an external signal (via the GPCR domain) that, via PIPK, directly activates downstream phospholipid signaling. Here we reveal the sporadic occurrence of GPCR-PIPKs in all eukaryotic supergroups, except for plants. Notably, all species having GPCR-PIPKs are unicellular microorganisms that favor aquatic environments. Phylogenetic analysis revealed that GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. In addition to GPCR-PIPKs, we identified five hitherto-unknown classes of GPCRs with accessory domains, four of which are universal players in signal transduction. Similarly to GPCR-PIPKs, this enables a direct coupling between extracellular sensing and downstream signaling. Overall, our findings point to an ancestral signaling system in eukaryotes where GPCR-mediated sensing is directly linked to downstream responses., IMPORTANCE G-protein-coupled receptors (GPCRs) are central sensors that activate eukaryotic signaling and are the primary targets of human drugs. In this report, we provide evidence for the widespread though limited presence of a novel class of GPCRs in a variety of unicellular eukaryotes. These include free-living organisms and organisms that are pathogenic for plants, animals, and humans. The novel GPCRs have a C-terminal phospholipid kinase domain, pointing to a direct link between sensing external signals via GPCRs and downstream intracellular phospholipid signaling. Genes encoding these receptors were likely present in the last common eukaryotic ancestor and were lost during the evolution of higher eukaryotes. We further describe five other types of GPCRs with a catalytic accessory domain, the so-called GPCR-bigrams, four of which may potentially have a role in signaling. These findings shed new light onto signal transduction in microorganisms and provide evidence for alternative eukaryotic signaling pathways.

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.

The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG-01131, PITG-01132, and PITG-16135, here denoted Piacwp1-3. Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.

The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2383-y) contains supplementary material, which is available to authorized users.

Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.

Recently, we provided the first genetic evidence for the requirement of tomato PLC4 and PLC6 genes in defense activation and disease resistance. The encoded enzymes were catalytically active as they were able to degrade phosphatidylinositol (PI), thereby producing diacylglycerol (DG). Here we report differential PLC gene expression following the initiation of defense signaling by the interaction between Cladosporium fulvum resistance (R) protein Cf-4 and its matching effector Avr4 in tomato hybrid seedlings that express both Cf-4 and Avr4. Furthermore, we observed that PLC3 and PLC6 gene expression is upregulated by elevated temperature in the control seedlings. This upregulation coincides with an increase in the levels of phosphatidic acid (PA) and a decrease in the levels of PI and phosphatidylinositol phosphate (PIP). The decrease in PI and PIP levels matches with the activation of PLC. In addition, the levels of the structural phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined transiently during recovery after the exposure to elevated temperature., Further studies will be required to explain the mechanism causing the sustained accumulation of PA during recovery, combined with a reduction in the levels of structural phospholipids.

The fungus Fusarium oxysporum f.sp. cubense (Focub) causes Fusarium wilt of banana. Focub strains are divided into races according to their host specificity, but which virulence factors underlie these interactions is currently unknown. In the F. oxysporum f.sp. lycopersici (Fol)-tomato system, small secreted fungal proteins, called Six proteins, were identified in the xylem sap of infected plants. The Fol Six1 protein contributes to virulence and has an avirulence function by activating the I-3 immune receptor of tomato. The Focub tropical race 4 (TR4) genome harbors three SIX1 homologs: SIX1a, b and c. In this study, the role of Focub-SIX1a in pathogenicity was evaluated since this homolog is present in not only TR4 but also in other races. A deletion mutant of the SIX1a gene from Focub TR4 strain II5 was generated (FocubΔSIX1a) and tested in planta. Mutants were found to be severely compromised in their virulence. Ectopic integration of the Focub-SIX1a gene in the FocubΔSIX1a strain restored virulence to wild type levels. We conclude that Focub-SIX1a is required for full virulence of Focub TR4 towards Cavendish banana.

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.

BACKGROUND: The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al. 2011. Science). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5,579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six nonribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third LP, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.

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 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.

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.

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.