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Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.

Ubiquitin-like domain-containing proteins (UDPs) are involved in the ubiquitin-proteasome system because of their ability to interact with the 26S proteasome. Here, we identified potato StUDP as a target of the Phytophthora infestans RXLR effector Pi06432 (PITG_06432), which supresses the salicylic acid (SA)-related immune pathway. By overexpressing and silencing of StUDP in potato, we show that StUDP negatively regulates plant immunity against P. infestans. StUDP interacts with, and destabilizes, the 26S proteasome subunit that is referred to as REGULATORY PARTICLE TRIPLE-A ATP-ASE (RPT) subunit StRPT3b. This destabilization represses the proteasome activity. Proteomic analysis and Western blotting show that StUDP decreases the stability of the master transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) in SA biosynthesis. StUDP negatively regulates the SA signalling pathway by repressing the proteasome activity and destabilizing StSARD1, leading to a decreased expression of the SARD1-targeted gene ISOCHORISMATE SYNTHASE 1 and thereby a decrease in SA content. Pi06432 stabilizes StUDP, and it depends on StUDP to destabilize StRPT3b and thereby supress the proteasome activity. Our study reveals that the P. infestans effector Pi06432 targets StUDP to hamper the homeostasis of the proteasome by the degradation of the proteasome subunit StRPT3b and thereby suppresses SA-related immunity.

Phytophthora species are devastating filamentous plant pathogens that belong to oomycetes, a group of microorganisms similar to fungi in morphology but phylogenetically distinct. They are sterol auxotrophic, but nevertheless exploit exogenous sterols for growth and development. However, as for now the mechanisms underlying sterol utilization in Phytophthora are unknown. In this study, we identified four genes in Phytophthora capsici that encode proteins containing a sterol-sensing domain (SSD), a protein domain of around 180 amino acids comprising five transmembrane segments and known to feature in sterol signaling in animals. Using a modified CRISPR/Cas9 system, we successfully knocked out the four genes named PcSCP1 to PcSCP4 (for P. capsici SSD-containing protein 1 to 4), either individually or sequentially, thereby creating single, double, triple, and quadruple knockout transformants. Results showed that knocking out just one of the four PcSCPs was not sufficient to block sterol signaling. However, the quadruple “all-four” PcSCPs knockout transformants no longer responded to sterol treatment in asexual reproduction, in contrast to wild-type P. capsici that produced zoospores under sterol treatment. Apparently, the four PcSCPs play a key role in sterol signaling in P. capsici with functional redundancy. Transcriptome analysis indicated that the expression of a subset of genes is regulated by exogenous sterols via PcSCPs. Further investigations showed that sterols could stimulate zoospore differentiation via PcSCPs by controlling actin-mediated membrane trafficking. Moreover, the pathogenicity of the “all-four” PcSCPs knockout transformants was significantly decreased and many pathogenicity related genes were downregulated, implying that PcSCPs also contribute to plant-pathogen interaction.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR–RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a ‘brute-force’ approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation ‘naifu’ invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

Filamentous plant pathogens apply mechanical forces to pierce their hosts surface and penetrate its tissues. DevastatingPhytophthorapathogens harness a specialized form of invasive tip growth to slice through the plant surface, wielding their hypha as a microscopic knife. Slicing requires a sharp hyphal tip that is not blunted at the site of the mechanical interaction. How tip shape is controlled, however, is unknown. We uncover an actin-based mechanostat inPhytophthora infestansthat controls tip sharpness during penetration. Mechanical stimulation of the hypha leads to the emergence of an aster-like actin configuration, which shows fast, local, and quantitative feedback to the local stress. We evidence that this functions as an adaptive mechanical scaffold that sharpens the invasive weapon and prevents it from blunting. The hyphal tip mechanostat enables the efficient conversion of turgor into localized invasive pressures that are required to achieve host penetration.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labeled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labeled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

Most potato cultivars are susceptible to late blight disease caused by the oomycete pathogen Phytophthora infestans. Here we report that the genetic loss of host susceptibility is a new source of resistance to prevent or diminish pathogen infection. Previously, we showed that RNAi-mediated silencing of the potato susceptibility (S) genes StDND1, StDMR1, and StDMR6 leads to increased late blight resistance. The mechanisms underlying this S-gene-mediated resistance have thus far not been identified. In this study, we examined the infection process of P. infestans in StDND1-, StDMR1-, and StDMR6-silenced potato lines. Microscopic analysis showed that penetration of P. infestans spores was hampered in StDND1-silenced plants. In StDMR1- and StDMR6-silenced plants, P. infestans infection was arrested at a primary infection stage by enhanced cell death responses. Histochemical staining revealed that StDMR1- and StDMR6-silenced plants display elevated ROS levels in cells at the infection sites. Resistance in StDND1-silenced plants, however, seems not to rely on a cell death response as ROS accumulation was found to be absent at most inoculated sites. Quantitative analysis of marker gene expression suggests that the increased resistance observed in StDND1- and StDMR6-silenced plants relies on an early onset of salicylic acid- and ethylene-mediated signaling pathways. Resistance mediated by silencing StDMR1 was found to be correlated with the early induction of salicylic acid-mediated signaling. These data provide evidence that different defense mechanisms are involved in late blight resistance mediated by functional impairment of different potato S-genes.

Phytophthora infestans causes late blight, a devastating disease of potato and tomato. Previous work in the Netherlands showed that the population is highly diverse due to sexual reproduction and that oospores can overwinter in the soil. We sequenced archived DNA from multiple nuclear and mitochondrial loci from P. infestans sampled in 1994 from 17 conventional and 5 organic potato fields and 7 potato refuse pile sites in the Southern Flevoland. We assessed the genealogical history using sequences from two regions of the single-copy nuclear ras gene (intron 1 and exons 3–6) and several mitochondrial loci (P3-rpl14, rpl5 and tRNAs and P4-cox1) from 83 isolates. There were 10 heterozygous sites in the ras gene and 12 in the mitochondrial genes that were phylogenetically informative. Five nuclear and 5 mitochondrial haplotypes were shared among fields and several unique haplotypes were found. Nucleotide diversity and population mean mutation rates (θW) were higher in samples from refuse piles and organic fields than conventional fields for both nuclear and mitochondrial loci. Subdivision was observed between conventional fields and refuse piles. Migration analysis suggested that gene flow may have occurred first from conventional potato fields to organic fields and then to refuse piles. Recent mutations were found in rare lineages from organic fields. Ancestral mutations in mitochondrial loci were derived from samples from refuse piles documenting the importance of refuse piles as a source of inoculum for late blight epidemics in the organic and conventional fields.

Sterols are a class of lipids with essential roles in sustaining the domain structure of cell membranes and regulating biological processes [1]. Human health is affected by high cholesterol levels [2]; drugs preventing this are widely used. Also striking is the high demand for compounds classified as sterol biosynthesis inhibitors (SBIs), either as medicine to control fungal infections or as agrochemicals to combat fungal plant diseases [3,4]. Despite the essential role of sterols in cell functioning in eukaryotes, several organisms, including nematodes, insects, and plasmodia, are not able to synthesize sterols themselves. To sustain normal development, these sterol auxotrophs may have to acquire exogenous sterols from their food or environment [5,6].

Late blight caused by the oomycete pathogen Phytophthora infestans is one of the most destructive diseases in potato cultivation. To successfully colonize its host, P. infestans secretes a suite of effector proteins that undermine plant immunity, many of which contain a conserved N-terminal RXLR motif that strongly resembles the host targeting motif in effectors of the malaria parasite Plasmodium falciparum. In this study, we focus on three P. infestans clade 5 aspartic proteases (PiAPs) that are homologous to Plasmepsin V (PMV), a Pl. falciparum AP responsible for cleaving effectors prior to translocation into red blood cells. Malaria parasites expressing mutated PMV are impaired in effector translocation and are less virulent. To determine whether clade 5 PiAPs play similar roles in virulence, we characterized P. infestans transformants with either reduced or enhanced PiAP expression levels. Phytophthora infestans transformants with altered PiAP10 or PiAP12 expression were found to be impaired in mycelial growth and sporangia production, and are hampered in their virulence on potato leaves. This was not observed in PiAP11 transformants. Activity assays showed that PiAP10 and PiAP12 possess moderate protease activity, and can potentially cleave the RXLR effector PiAVR4, but not a PiAVR4 version with a mutated RXLR motif. These findings imply that P. infestans APs function in the proteolytic cleavage of RXLR effectors, and warrant further investigation to verify and confirm the role of clade 5 PiAPs in effector processing.

Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying β-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between β-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity.

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases.

Metabolism is the set of biochemical reactions of an organism that enables it to assimilate nutrients from its environment and to generate building blocks for growth and proliferation. It forms a complex network that is intertwined with the many molecular and cellular processes that take place within cells. Systems biology aims to capture the complexity of cells, organisms, or communities by reconstructing models based on information gathered by high-throughput analyses (omics data) and prior knowledge. One type of model is a genome-scale metabolic model (GEM) that allows studying the distributions of metabolic fluxes, i.e., the “mass-flow” through the network of biochemical reactions. GEMs are nowadays widely applied and have been reconstructed for various microbial pathogens, either in a free-living state or in interaction with their hosts, with the aim to gain insight into mechanisms of pathogenicity. In this review, we first introduce the principles of systems biology and GEMs. We then describe how metabolic modeling can contribute to unraveling microbial pathogenesis and host–pathogen interactions, with a specific focus on oomycete plant pathogens and in particular Phytophthora infestans. Subsequently, we review achievements obtained so far and identify and discuss potential pitfalls of current models. Finally, we propose a workflow for reconstructing high-quality GEMs and elaborate on the resources needed to advance a system biology approach aimed at untangling the intimate interactions between plants and pathogens.

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, β, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1., Author summary Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, β, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In oomycete plant pathogen Phytophthora sojae, previous research showed that G protein α subunit is involved in zoospore motility and pathogenicity. However, the elaborate functions of the G protein α subunit remain largely unknown. Here, we identified a serine/threonine protein kinase, PsYPK1, which directly interacts with the G protein α subunit (PsGPA1) in P. sojae. We found that PsYPK1 regulates the production of sporangia and oospores, mycelial growth in nutrient poor medium, and pathogenicity in P. sojae. We further revealed that PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and pathogenicity of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, accounting for the negative role of PsGPA1 in sporangium formation. Our study is the first to show that G protein signaling is transmitted to a downstream effector to regulate the development and pathogenicity of an oomycete pathogen, which opens new avenues for researches on animal and plant pathogens and host-microbe interactions.

The de novo biosynthesis of sterols is critical for the majority of eukaryotes; however, some organisms lack this pathway, including most oomycetes. Phytophthora spp. are sterol auxotrophic but, remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here, we show that PcDHCR7, a gene in Phytophthora capsici predicted to encode Δ7-sterol reductase, displays multiple functions. When expressed in Saccharomyces cerevisiae , PcDHCR7 showed the Δ7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has the Δ7-sterol reductase activity in P. capsici itself . This enables P. capsici to transform sterols recruited from the environment for better use. The biological characteristics of ΔPcDHCR7 transformants were compared with those of the wild-type strain and a PcDHCR7 complemented transformant, and the results showed that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores. Further analysis of the transcriptome indicated that the expression of many genes changed in the ΔPcDHCR7 transformant, which involve in different biological processes. It is possible that P. capsici compensates for the defects caused by the loss of PcDHCR7 by remodelling its transcriptome.

The oomycete Phytophthora infestans is a notorious plant pathogen with potato and tomato as its primary hosts. Previous research showed that the heterotrimeric G-protein subunits Gα and Gβ have a role in zoospore motility and virulence, and sporangial development, respectively. Here, we present analyses of the gene encoding a Gγ subunit in P. infestans, Pigpg1. The overall similarity of PiGPG1 with non-oomycete Gγ subunits is low, with only the most conserved amino acids maintained, but similarity with its homologs in other oomycetes is high. Pigpg1 is expressed in all life stages and shows a similar expression profile as the gene encoding the Gβ subunit, Pigpb1. To elucidate its function, transformants were generated in which Pigpg1 is silenced or overexpressed and their phenotypes were analyzed. Pigpg1-silenced lines produce less sporangia, which are malformed. Altogether, the results show that PiGPG1 is crucial for proper sporangia development and zoosporogenesis. PiGPG1 is a functional Gγ, and likely forms a dimer with PiGPB1 that mediates signaling., Fungal Genetics and Biology, 116, ISSN:1087-1845, ISSN:1096-0937

This study provides insight into the evolution of the exocyst subunits of Solanaceous plants and is the first to show their role in immunity against multiple unrelated pathogens., The exocyst, a multiprotein complex consisting of eight subunits, plays an essential role in many biological processes by mediating secretion of post-Golgi-derived vesicles towards the plasma membrane. In recent years, roles for plant exocyst subunits in pathogen defence have been uncovered, largely based on studies in the model plant Arabidopsis. Only a few studies have been undertaken to assign the role of exocyst subunits in plant defence in other plants species, including crops. In this study, predicted protein sequences from exocyst subunits were retrieved by mining databases from the Solanaceous plants Nicotiana benthamiana, tomato, and potato. Subsequently, their evolutionary relationship with Arabidopsis exocyst subunits was analysed. Gene silencing in N. benthamiana showed that several exocyst subunits are required for proper plant defence against the (hemi-)biotrophic plant pathogens Phytophthora infestans and Pseudomonas syringae. In contrast, some exocyst subunits seem to act as susceptibility factors for the necrotrophic pathogen Botrytis cinerea. Furthermore, the majority of the exocyst subunits were found to be involved in callose deposition, suggesting that they play a role in basal plant defence. This study provides insight into the evolution of exocyst subunits in Solanaceous plants and is the first to show their role in immunity against multiple unrelated pathogens.

Background The oomycete Phytophthora infestans causes late blight on potato and tomato. Despite extensive research, the P. infestans-host interaction is still poorly understood. To find new ways to further unravel this interaction we established a new infection system using MsK8 tomato cells. These cells grow in suspension and can be maintained as a stable cell line that is representative for tomato. Results MsK8 cells can host several Phytophthora species pathogenic on tomato. Species not pathogenic on tomato could not infect. Microscopy revealed that 16 h after inoculation up to 36% of the cells were infected. The majority were penetrated by a germ tube emerging from a cyst (i.e. primary infection) while other cells were already showing secondary infections including haustoria. In incompatible interactions, MsK8 cells showed defense responses, namely reactive oxygen species production and cell death leading to a halt in pathogen spread at the single cell level. In compatible interactions, several P. infestans genes, including RXLR effector genes, were expressed and in both, compatible and incompatible interactions tomato genes involved in defense were differentially expressed. Conclusions Our results show that P. infestans can prosper as a pathogen in MsK8 cells; it not only infects, but also makes haustoria and sporulates, and it receives signals that activate gene expression. Moreover, MsK8 cells have the ability to support pathogen growth but also to defend themselves against infection in a similar way as whole plants. An advantage of MsK8 cells compared to leaves is the more synchronized infection, as all cells have an equal chance of being infected. Moreover, analyses and sampling of infected tissue can be performed in a non-destructive manner from early time points of infection onwards and as such the MsK8 infection system offers a potential platform for large-scale omics studies and activity screenings of inhibitory compounds.

The oomycete pathogen Phytophthora infestans causes potato and tomato late blight, a disease that is a serious threat to agriculture. P. infestans is a hemibiotrophic pathogen, and during infection, it scavenges nutrients from living host cells for its own proliferation. To date, the nutrient flux from host to pathogen during infection has hardly been studied, and the interlinked metabolisms of the pathogen and host remain poorly understood. Here, we reconstructed an integrated metabolic model of P. infestans and tomato (Solanum lycopersicum) by integrating two previously published models for both species. We used this integrated model to simulate metabolic fluxes from host to pathogen and explored the topology of the model to study the dependencies of the metabolism of P. infestans on that of tomato. This showed, for example, that P. infestans, a thiamine auxotroph, depends on certain metabolic reactions of the tomato thiamine biosynthesis. We also exploited dual-transcriptome data of a time course of a full late blight infection cycle on tomato leaves and integrated the expression of metabolic enzymes in the model. This revealed profound changes in pathogen-host metabolism during infection. As infection progresses, P. infestans performs less de novo synthesis of metabolites and scavenges more metabolites from tomato. This integrated metabolic model for the P. infestans-tomato interaction provides a framework to integrate data and generate hypotheses about in planta nutrition of P. infestans throughout its infection cycle. IMPORTANCE Late blight disease caused by the oomycete pathogen Phytophthora infestans leads to extensive yield losses in tomato and potato cultivation worldwide. To effectively control this pathogen, a thorough understanding of the mechanisms shaping the interaction with its hosts is paramount. While considerable work has focused on exploring host defense mechanisms and identifying P. infestans proteins contributing to virulence and pathogenicity, the nutritional strategies of the pathogen are mostly unresolved. Genome-scale metabolic models (GEMs) can be used to simulate metabolic fluxes and help in unravelling the complex nature of metabolism. We integrated a GEM of tomato with a GEM of P. infestans to simulate the metabolic fluxes that occur during infection. This yields insights into the nutrients that P. infestans obtains during different phases of the infection cycle and helps in generating hypotheses about nutrition in planta.

One century ago, on February 10, 1917, Johanna Westerdijk delivered her inaugural speech at Utrecht University entitled “New directions in phytopathological research”. By doing so, she became the very first female professor in the Netherlands and set the stage for many female professors who followed her example. Besides her remarkable performance as a role model for women in science, Johanna Westerdijk was also a pioneer in the global science field of phytopathology, which rapidly emerged at the end of the 19th century and reached maturity during the course of her scientific career.

Key message Transgenic Nicotiana benthamiana lines with constitutive expression of an Arabidopsis lectin receptor kinase gene ( LecRK - I.9 or LecRK - IX.1 ) show enhanced resistance to Phytophthora pathogens, demonstrating conserved gene functionality after interfamily transfer. Abstract In plants, cell surface receptors mediate the first layer of innate immunity against pathogenic microbes. In Arabidopsis several L-type lectin receptor kinases (LecRKs) were previously found to function as Phytophthora resistance components. In this study, we determined the functionality of Arabidopsis LecRK-I.9 or LecRK-IX.1 in Phytophthora resistance when transferred into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines were generated for each LecRK gene and molecular analyses revealed variation in transgene copy number, transgene expression levels and LecRK protein accumulation. Infection assays showed that transgenic N. benthamiana plants expressing either Arabidopsis LecRK-I.9 or LecRK-IX.1 are more resistant to Phytophthora capsici and to Phytophthora infestans. These results demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora resistance function when transferred into N. benthamiana. Therefore, these LecRKs have the potential to function as a complementary Phytophthora resistance resource in distantly related plant species next to the canonical Phytophthora resistance genes encoding nucleotide-binding leucine-rich repeat proteins.

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.

Late blight disease caused by the plant pathogenic oomycete pathogen Phytophthora infestans is one of the most limiting factors in potato production. P. infestans is able to overcome introgressed late blight resistance by adaptation of effector genes. AVR1 is an RXLR effector that triggers immune responses when recognized by the potato resistance protein R1. P. infestans isolates avirulent on R1 plants were found to have AVR1 variants that are recognized by R1. Virulent isolates though, lack AVR1 but do contain a close homologue of AVR1, named A-L, of which all variants escape recognition by R1. Co-expression of AVR1 and R1 in Nicotiana benthamiana results in a hypersensitive response (HR). In contrast, HR is not activated when A-L is co-expressed with R1. AVR1 and A-L are highly similar in structure. They share two W motifs and one Y motif in the C-terminal part but differ in the T-region, a 38 amino acid extension at the carboxyl-terminal tail of AVR1 lacking in A-L. To pinpoint what determines R1-mediated recognition of AVR1 we tested elicitor activity of AVR1 and A-L chimeric and deletion constructs by co-expression with R1. The T-region is important as it enables R1-mediated recognition of A-L, not only when fused to A-L but also via trans-complementation. Yet, AVR1 lacking the T-region is still active as an elicitor of HR, but this activity is lost when certain motifs are swapped with A-L. These data show that A-L circumvents R1 recognition not only because it lacks the T-region, but also because of differences in the conserved C-terminal effector motifs.

Oomycetes are diverse eukaryotic microorganisms, comprising important pathogens as well as free-living saprophytes [1]. Plant pathogenic oomycetes cause enormous yield losses in crop plants but also threaten natural vegetation. Phytophthora infestans, the causal agent of potato late blight, is without a doubt the most (in)famous. Other important plant pathogenic oomycetes include downy mildews and Albugo [2]. Animal pathogenic oomycetes (such as Saprolegnia, Aphanomyces, and Pythium) primarily affect marine organisms such as fish and crustaceans but can also cause harmful diseases in humans and cattle. Here, we outline unique features of oomycetes and their cellular signaling systems. We focus on one particularly interesting class of signaling components, the so-called GPCR-bigrams, in which a typical signaling domain is preceded by a G-protein coupled receptor (GPCR) domain and propose future directions to elucidate the function and activity of this exceptional group of signaling proteins., PLoS Pathogens, 14 (7), ISSN:1553-7374, ISSN:1553-7366

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.

Members of the family of 70-kD heat shock proteins (HSP70 s) play various stress-protective roles in plants. In this study, a wheat HSP70 gene was isolated from a suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis f. sp. tritici. The gene, that was designated as TaHSC70, was predicted to encode a protein of 690 amino acids, with a molecular mass of 73.54 KDa and a pI of 5.01. Further analysis revealed the presence of a conserved signature that is characteristic for HSP70s and phylogenetic analysis demonstrated that TaHSC70 is a homolog of chloroplast HSP70s. TaHSC70 mRNA was present in leaves of both green and etiolated wheat seedlings and in stems and roots. The transcript level in roots was approximately threefold less than in leaves but light–dark treatment did not charge TaHSC70 expression. Following heat shock of wheat seedlings at 40°C, TaHSC70 expression increased in leaves of etiolated seedlings but remained stable at the same level in green seedlings. In addition, TaHSC70 was differentially expressed during an incompatible and compatible interaction with wheat-stripe rust, and there was a transient increase in expression upon treatment with methyl jasmonate (MeJA) treatment. Salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) treatments had no influence on TaHSC70 expression. These results suggest that TaHSC70 plays a role in stress-related responses, and in defense responses elicited by infection with stripe rust fungus and does so via a JA-dependent signal transduction pathway.

Introgression breeding has resulted in several potato lines that are resistant to late blight, a devastating plant disease caused by the oomycete Phytophthora infestans. The traditional differential set consists of potato lines with 11 late blight resistance specificities, referred to as R1 to R11. With the exception of the R4 locus, all the resistance loci in these lines have been genetically mapped or positioned in resistance (R) gene clusters. In this study, we show that potato lines that are defined to carry R4 do not necessarily recognize the same P. infestans strains. Field isolates appeared to be avirulent on either the R4 differential developed by Mastenbroek or the one developed by Black but not on both. Previously, we identified the avirulence gene PiAvr4, which is a member of the RXLR effector family. In planta expression of PiAvr4 revealed that recognition of PiAvr4 is strictly confined to the Mastenbroek R4 differential. Segregation of the trait in two independent F1 progenies showed that late blight resistance in this differential is determined by a single dominant gene, now referred to as R4Ma.

SUMMARY Oomycete RXLR-dEER effector proteins are rapidly evolving proteins with the selective pressure targeted predominantly at their C-terminal ends. The majority of RXLR-dEER proteins have recognizable motifs of 21-30 amino acids in the C-terminal domain that are named after conserved amino acid residues at fixed positions within the respective motifs. In this article, it is reported that the Phytophthora infestans RXLR-dEER protein Avr4 contains three W motifs and one Y motif in its C-terminal domain. Agroinfection assays using constructs encoding modified forms of PiAvr4 have shown that the region containing the W2 motif, in combination with either the W1 or W3 motif, triggers a necrotic response in potato plants carrying the resistance gene R4. By mining the superfamily of avirulence homologues (Avh) deduced from three sequenced Phytophthora genomes, several Avh proteins were identified as homologues of PiAvr4: six in P. infestans, one in P. ramorum and seven in P. sojae. One very close homologue of PiAvr4 was cloned from the sibling species, P. mirabilis. This species is not pathogenic on potato but, similar to PiAvr4, PmirAvh4 triggered a necrotic response on potato clones carrying R4, but not on clones lacking R4. Genes encoding RXLR-dEER effectors are often located in regions showing genome rearrangements. Alignment of the genomic region harbouring PiAvr4 with syntenic regions in P. sojae and P. ramorum revealed that PiAvr4 is located on a 100-kb indel block and is surrounded by transposable elements.

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1–mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1–mediated resistance in potato.

Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens , has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein α subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling.

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.

Resistance in potato against the oomycete Phytophthora infestans is conditioned by resistance (R) genes that are introgressed from wild Solanum spp. into cultivated potato. According to the gene-for-gene model, proteins encoded by R genes recognize race-specific effectors resulting in a hypersensitive response (HR). We isolated P. infestans avirulence gene PiAvr4 using a combined approach of genetic mapping, transcriptional profiling, and bacterial artificial chromosome marker landing. PiAvr4 encodes a 287-amino-acid-protein that belongs to a superfamily of effectors sharing the putative host-cell-targeting motif RXLR-dEER. Transformation of P. infestans race 4 strains with PiAvr4 resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Moreover, expression of PiAvr4 in R4 plants using PVX agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 but not r0 plants. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4. This was confirmed in agroinfiltration assays but not with PVX agroinfection. Because there was always recognition of PiAvr4 retaining the signal peptide, extracellular recognition cannot be excluded. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in PiAvr4 that result in truncated peptides; hence, PiAvr4 is apparently not crucial for virulence.

For the soybean pathogen Phytophthora sojae , chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1 , is a single-copy gene in P. sojae and encodes a G-protein α subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans . To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1 -silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1 , and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4 , were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.

The oomycete Phytophthora infestans is a filamentous plant pathogen that causes the late blight disease in potato worldwide. It has been a favorite subject of study since the Great Irish Famine in the 1840s and is considered to be a model species for oomycetes. Its genome of over 240 Mb has a remarkable organization with gene dense regions interspersed with gene poor regions, the latter harboring genes involved in host specificity and virulence. These genes are key players in the arms race with the host. They can easily mutate to avoid recognition by immune receptors and their abundance shows that P. infestans possesses an impressive arsenal of weapons for attacking potato and is likely hard to beat.

The comparative analysis of plant gene families in a phylogenetic framework has greatly accelerated due to advances in next generation sequencing. In this study, we provide an evolutionary analysis of the L-type lectin receptor kinase and L-type lectin domain proteins (L-type LecRKs and LLPs) that are considered as components in plant immunity, in the plant family Brassicaceae and related outgroups. We combine several lines of evidence provided by sequence homology, HMM-driven protein domain annotation, phylogenetic analysis, and gene synteny for large-scale identification of L-type LecRK and LLP genes within nine core-eudicot genomes. We show that both polyploidy and local duplication events (tandem duplication and gene transposition duplication) have played a major role in L-type LecRK and LLP gene family expansion in the Brassicaceae. We also find significant differences in rates of molecular evolution based on the mode of duplication. Additionally, we show that LLPs share a common evolutionary origin with L-type LecRKs and provide a consistent gene family nomenclature. Finally, we demonstrate that the largest and most diverse L-type LecRK clades are lineage-specific. Our evolutionary analyses of these plant immune components provide a framework to support future plant resistance breeding.

The year 2004 was an exciting one for the Phytophthora research community. The United States Department of Energy Joint Genome Institute (JGI) completed the draft genome sequence of two Phytophthora species, Phytophthora sojae and Phytophthora ramorum. In August of that year over 50 people gathered at JGI in Walnut Creek, California, for an annotation jamboree and searched for the secrets and surprises that the two genomes have in petto. This culminated in a paper in Science in September of this year describing the highlights of the sequencing project and emphasizing the power of having the genome sequences of two closely related organisms. This MPMI Focus issue on Phytophthora genomics contains a number of more specialized manuscripts centered on gene annotation and genome organization, and complemented with manuscripts that rely on genomics resources.

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.

Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of P. ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements also have occurred. Deletions and duplications often were found in association with genes encoding secreted proteins, including effectors that are important for interaction with host plants. Among secreted protein genes, different evolutionary patterns were found. Elicitin genes that code for a complex family of highly conserved Phytophthora-specific elicitors show conservation in gene number and order, and often are clustered. In contrast, the race-specific elicitor gene Avr1b-1 appeared to be missing from the region with conserved synteny, as were its five homologs that are scattered over the four genomic regions. Some gene families encoding secreted proteins were found to be expanded in one species compared with the other. This could be the result of either repeated gene duplications in one species or specific deletions in the other. These different evolutionary patterns may shed light on the functions of these secreted proteins in the biology and pathology of the two Phytophthora spp.

The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes. It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans , as was concluded from previous studies on the role of the G-protein α-subunit PiGPA1 in this organism. The expression of the P. infestans G-protein β-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formation. The gene was hardly expressed in mycelium incubated in rich growth medium. The introduction of additional copies of Pigpb1 into the genome led to silencing of the gene and resulted in transformants deficient in PiGPB1. These Pigpb1 -silenced mutants formed very few asexual spores (sporangia) when cultured in rye sucrose medium and produced a denser mat of aerial mycelium than the wild type. Partially Pigpb1 -silenced mutants showed intermediate phenotypes with regard to sporulation, and a relatively large number of their sporangia were malformed. The results show that PiGPB1 is important for vegetative growth and sporulation and, therefore, for the pathogenicity of this organism.

An 85-kDa beta-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) beta-D-glucopyranoside and, to lesser extent, towards 4-MU-D-xylopyranoside, but not towards 4-MU-beta-D-glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.

A G protein alpha subunit gene (pigpa1) and a G protein beta subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of alpha,beta, and gamma subunits and participate in diverse signal transduction pathways. The deduced amino acid sequence of both pigpa1 and pigpb1, showed the typical conserved motifs present in Galpha or Gbeta proteins from other eukaryotes. Southern blot analysis revealed no additional copies of Galpha or Gbeta subunit genes in P. infestans, suggesting that pigpa1 and pigpb1 are single copy genes. By cross-hybridization homologues of gpa1 and gpb1 were detected in other Phythophthora species. Expression analyses revealed that both genes are differentially expressed during asexual development, with the highest mRNA levels in sporangia. In mycelium, no pigpa1 mRNA was detected. Western blot analysis using a polyclonal GPA1 antibody confirmed the differential expression of pigpa1. These expression patterns suggest a role for G-protein-mediated signaling during formation and germination of asexual spores of P. infestans, developmental stages representing the initial steps of the infection process.

Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security1,2. All late blight resistance genes identified to date belong to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors3. However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors4. Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.Potato (Solanum tuberosum L.) is the most important non-grain food crop and a major source of calories for the world's poor5. Increasing potato production is critical to prevent global malnutrition and hunger in an era of expanding world population. Unfortunately, potato suffers from the devastating late blight disease, which is caused by the notorious oomycete pathogen Phytophthora infestans. To limit losses to late blight, potato breeders rely on fungicide treatment and breeding of disease resistance (R) genes, all of which identified thus far belong to the coiled-coil, nucleotide-binding, leucine-rich repeat (CC-NB-LRR) class of immune receptors. These intracellular proteins recognize pathogen avirulence (Avr) proteins of the RxLR class of effectors to mount defence responses. However, RxLR effectors display high evolutionary rates4, and as a result, P. infestans can rapidly circumvent recognition by intracellular R immune receptors, thereby limiting the development of sustainable and durable genetic resistance. Therefore, novel types of immune receptors that recognize a broader spectrum of pathogen molecules are needed.To fend off pathogens, plants rely on two classes of immune receptors that either reside inside the plant cell (NB-LRRs) or on the cell surface. The first line of defence is initiated by surface receptors, also called pattern recognition receptors (PRRs). PRRs are characteristically receptor-like proteins (RLPs), such as Ve1/Ve26, Cfs7,8 and LeEIX19, or receptor-like kinases (RLKs), such as FLS2, EFR and XA2110. PRRs typically recognize conserved pathogen-associated molecular patterns (PAMPs)11. So far, only a relatively few cell surface receptors against agronomically important pathogens have been identified.Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium pathogen species (Pfam PF00964)12,​13,​14. P. infestans contains six elicitin genes that are conserved among different strains13,15. Elicitins are recognized as oomycete PAMPs but their intrinsic function in oomycetes is to bind lipids. Some elicitins sequester sterols from plants, thereby fulfilling an important biological function in Phytophthora and Pythium species that cannot synthesize sterols12. Targeting such conserved ‘Achilles heel’ proteins of pathogens is expected to lead to a more broad-spectrum resistance.To identify novel types of potential immune receptors against the potato late blight pathogen, we initiated the cloning of ELR, a gene that determines response to elicitins16. We screened a collection of wild Solanum germplasm by Potato virus X (PVX) agroinfection for responses to INF1, a secreted elicitin of P. infestans (Fig. 1a). Solanum microdontum genotype mcd360-1 consistently responded to INF1 with a cell death response (Fig. 1b). We crossed mcd360-1 with S. microdontum ssp. gigantophyllum gig714-1 that does not respond to INF1. The F1 population segregated for response to INF1 in a 1:1 ratio, which suggests that ELR is a single dominant gene.

Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.

Recent phylogenetic analyses of the nucleotide binding sites (NBS)-leucine-rich repeats (LRR) class of plant disease resistance (R) genes suggest that these genes are ancient and coexist next to susceptibility alleles at resistance loci. Another class of R genes encodes serine-threonine protein kinases related to Pto that were originally identified from wild relatives of tomato. In this study, we exploit the highly diverse genus Solanum to identify Pto-like sequences and test various evolutionary scenarios for Pto-like genes. Polymerase chain reaction amplifications with the use of primers that were developed on the basis of conserved and variable regions of Pto revealed an extensive Pto gene family and yielded 32 intact Pto-like sequences from six Solanum species. Furthermore, Pto-like transcripts were detected in the leaf tissue of all tested plants. The kinase consensus and autophosphorylation sites were highly conserved, in contrast to the kinase activation domain, which is involved in ligand recognition in Pto. Phylogenetic analyses distinguished nine classes of Pto-like genes and revealed that orthologs were more similar than paralogs, suggesting that the Pto gene family evolved through a series of ancient gene duplication events prior to speciation in Solanum. Thus, like the NBS-LRR class, the kinase class of R genes is highly diverse and ancient.