Trafficking arms: oomycete effectors enter host plant cells

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.     

Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.     

A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B

The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.     

Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death

Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors.     

Pathogen virulence of Phytophthora infestans: from gene to functional genomics

The oomycete, Phytophthora infestans, is one of the most important plant pathogens worldwide. Much of the pathogenic success of P. infestans, the potato late blight agent, relies on its ability to generate large amounts of sporangia from mycelia, which release zoospores that encyst and form infection structures. Until recently, little was known about the molecular basis of oomycete pathogenicity by the avirulence molecules that are perceived by host defenses. To understand the molecular mechanisms interplay in the pathogen and host interactions, knowledge of the genome structure was most important, which is available now after genome sequencing. The mechanism of biotrophic interaction between potato and P. infestans could be determined by understanding the effector biology of the pathogen, which is until now poorly understood. The recent availability of oomycete genome will help in understanding of the signal transduction pathways followed by apoplastic and cytoplasmic effectors for translocation into host cell. Finally based on genomics, novel strategies could be developed for effective management of the crop losses due to the late blight disease.     

Entry of oomycete and fungal effectors into plant and animal host cells

Fungal and oomycete pathogens cause many destructive diseases of plants and important diseases of humans and other animals. Fungal and oomycete plant pathogens secrete numerous effector proteins that can enter inside host cells to condition susceptibility. Until recently it has been unknown if these effectors enter via pathogen-encoded translocons or via pathogen-independent mechanisms. Here we review recent evidence that many fungal and oomycete effectors enter via receptor-mediated endocytosis, and can do so in the absence of the pathogen. Surprisingly, a large number of these effectors utilize cell surface phosphatidyinositol-3-phosphate (PI-3-P) as a receptor, a molecule previously known only inside cells. Binding of effectors to PI-3-P appears to be mediated by the cell entry motif RXLR in oomycetes, and by diverse RXLR-like variants in fungi. PI-3-P appears to be present on the surface of animal cells also, suggesting that it may mediate entry of effectors of fungal and oomycete animal pathogens, for example, RXLR effectors found in the oomycete fish pathogen, Saprolegnia parasitica. Reagents that can block PI-3-P-mediated entry have been identified, suggesting new therapeutic strategies.     

Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.     

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.     

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.     

Molecular Profiling of the Phytophthora plurivora Secretome: A Step towards Understanding the Cross-Talk between Plant Pathogenic Oomycetes and Their Hosts

The understanding of molecular mechanisms underlying host–pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.     

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

Groovy times: filamentous pathogen effectors revealed

Filamentous microorganisms, such as fungi and oomycetes, secrete an arsenal of effector proteins that modulate plant innate immunity and enable parasitic infection. Deciphering the biochemical activities of effectors to understand how pathogens successfully colonize and reproduce on their host plants became a driving paradigm in the field of fungal and oomycete pathology. Recent findings illustrate a diversity of effector structures and activities, as well as validate the view that effector genes are the target of the evolutionary forces that drive the antagonistic interplay between pathogen and host.     

Recent developments in effector biology of filamentous plant pathogens

Filamentous pathogens, such as plant pathogenic fungi and oomycetes, secrete an arsenal of effector molecules that modulate host innate immunity and enable parasitic infection. It is now well accepted that these effectors are key pathogenicity determinants that enable parasitic infection. In this review, we report on the most interesting features of a representative set of filamentous pathogen effectors and highlight recent findings. We also list and describe all the linear motifs reported to date in filamentous pathogen effector proteins. Some of these motifs appear to define domains that mediate translocation inside host cells.     

The malarial host-targeting signal is conserved in the Irish potato famine pathogen

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within approximately 25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of approximately 25-30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of approximately 30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens.     

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.     

Pathogen effectors: What do they do at plasmodesmata?

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.