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The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis (Arabidopsis thaliana) initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-mediated adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying the tolerance of Arabidopsis to cyst nematode infections.

Nematode migration, feeding site formation, withdrawal of plant assimilates, and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognized as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana, since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode–plant interactions. Through imaging of tolerance-related parameters, the green canopy area was identified as an accessible and robust measure for assessing damage due to cyst nematode infection. Subsequently, a high-throughput phenotyping platform simultaneously measuring the green canopy area growth of 960 A. thaliana plants was developed. This platform can accurately measure cyst nematode and root-knot nematode tolerance limits in A. thaliana through classical modelling approaches. Furthermore, real-time monitoring provided data for a novel view of tolerance, identifying a compensatory growth response. These findings show that our phenotyping platform will enable a new mechanistic understanding of tolerance to below-ground biotic stress.

Plant root architecture plasticity in response to biotic stresses has not been thoroughly investigated. Infection by endoparasitic cyst nematodes induces root architectural changes that involve the formation of secondary roots at infection sites. However, the molecular mechanisms regulating secondary root formation in response to cyst nematode infection remain largely unknown. We first assessed whether secondary roots form in a nematode density-dependent manner by challenging wild-type Arabidopsis plants with increasing numbers of cyst nematodes (Heterodera schachtii). Next, using jasmonate-related reporter lines and knockout mutants, we tested whether tissue damage by nematodes triggers jasmonate-dependent secondary root formation. Finally, we verified whether damage-induced secondary root formation depends on local auxin biosynthesis at nematode infection sites. Intracellular host invasion by H. schachtii triggers a transient local increase in jasmonates, which activates the expression of ERF109 in a COI1-dependent manner. Knockout mutations in COI1 and ERF109 disrupt the nematode density-dependent increase in secondary roots observed in wild-type plants. Furthermore, ERF109 regulates secondary root formation upon H. schachtii infection via local auxin biosynthesis. Host invasion by H. schachtii triggers secondary root formation via the damage-induced jasmonate-dependent ERF109 pathway. This points at a novel mechanism underlying plant root plasticity in response to biotic stress.

To establish persistent infections in host plants, herbivorous invaders, such as root-knot nematodes, must rely on effectors for suppressing damage-induced jasmonate-dependent host defenses. However, at present, the effector mechanisms targeting the biosynthesis of biologically active jasmonates to avoid adverse host responses are unknown.Using yeast-two-hybrid, in planta co-immunoprecipitation, and mutant analyses, we identified 12-oxophytodienoate reductase 2 (OPR2) as an important host target of the stylet-secreted effector MiMSP32 of the root-knot nematode Meloidogyne incognita. MiMSP32 has no informative sequence similarities with other functionally annotated genes but was selected for the discovery of novel effector mechanisms based on evidence of positive, diversifying selection.OPR2 catalyzes the conversion of a derivative of 12-oxophytodienoate to jasmonic acid (JA) and operates parallel to 12-oxophytodienoate reductase 3 (OPR3), which controls the main pathway in the biosynthesis of jasmonates. We show that MiMSP32 targets OPR2 to promote parasitism of M. incognita in host plants independent of OPR3-mediated JA biosynthesis.Artificially manipulating the conversion of the 12-oxophytodienoate by OPRs increases susceptibility to multiple unrelated plant invaders. Our study is the first to shed light on a novel effector mechanism targeting this process to regulate susceptibility of host plants

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are essential for PCN control. However, the poor delineation of G. pallida pathotypes has hampered the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs (venom allergen-like proteins) diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCNs after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (“DOG boxes”) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with the expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Resequencing of PCN populations with different virulence characteristics will allow for the linking of these characteristics to the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

We compared the gene expression of wild-type Col-0 and a T-DNA mutant SALK_116381C (opr2-1). We either infected or mock-infected the plants with the root knot nematode Meloidogyne incognita and measured the root transcriptome after 0, 1, 4, and 7 days post infection using RNA-seq. The aim of the experiment was to determine whether opr2-1 affected gene expression patterns induced by nematode infection.

Cyst nematodes are considered a dominant threat to yield for a wide range of major food crops. Current control strategies are mainly dependent on crop rotation and the use of resistant cultivars. Various crops exhibit single dominant resistance (R) genes that are able to activate effective host-specific resistance to certain cyst nematode species and/or populations. An example is the potato R gene Gpa2, which confers resistance against the potato cyst nematode (PCN), Globodera pallida population D383. Activation of Gpa2 results in a delayed resistance response, which is characterized by a layer of necrotic cells formed around the developing nematode feeding structure. However, knowledge about the Gpa2-induced defense pathways is still lacking. Here, we uncover the transcriptional changes and gene expression network induced upon Gpa2 activation in potato roots infected with G. pallida. To this end, in vitro-grown Gpa2-resistant potato roots were infected with the avirulent population D383 and virulent population Rookmaker. Infected root segments were harvested at 3 and 6 dpi and sent for RNA sequencing. Comparative transcriptomics revealed a total of 1,743 differentially expressed genes (DEGs) upon nematode infection, of which 559 DEGs were specifically regulated in response to D383 infection. D383-specific DEGs associated with Gpa2-mediated defense mainly relates to calcium-binding activity, salicylic acid (SA) biosynthesis, and systemic acquired resistance (SAR). These data reveal that cyst nematode resistance in potato roots depends on conserved downstream signaling pathways involved in plant immunity, which are also known to contribute to R genes-mediated resistance against other pathogens with different lifestyles.

The activity of intracellular plant nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is therefore crucial to advance our understanding of how these receptors function. A co-immunoprecipitation/mass spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the resistance protein Gpa2, a CC-NB-LRR immune receptor conferring resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defense. A N. benthamiana homolog of the GLYCINE-RICH RNA-BINDING PROTEIN7 (NbGRP7) protein was prioritized as a Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif (RRM) compromises its role in Rx1-mediated defense. Strikingly, ectopic NbGRP7 expression is likely to impact the steady-state levels of Rx1, which relies on an intact RRM. Our findings illustrate that NbGRP7 is a pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 function at a posttranscriptional level.

Infections by root-feeding nematodes have profound effects on root system architecture and consequently shoot growth of host plants. Plants harbor intraspecific variation in their growth responses to belowground biotic stresses by nematodes, but the underlying mechanisms are not well understood. Here, we show that the transcription factor TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR-9 (TCP9) modulates root system architectural plasticity in Arabidopsis thaliana in response to infections by the endoparasitic cyst nematode Heterodera schachtii. Young seedlings of tcp9 knock-out mutants display a significantly weaker primary root growth inhibition response to cyst nematodes than wild-type Arabidopsis. In older plants, tcp9 reduces the impact of nematode infections on the emergence and growth of secondary roots. Importantly, the altered growth responses by tcp9 are most likely not caused by less biotic stress on the root system, because TCP9 does not affect the number of infections, nematode development, and size of the nematode-induced feeding structures. RNA-sequencing of nematode-infected roots of the tcp9 mutants revealed differential regulation of enzymes involved in reactive oxygen species (ROS) homeostasis and responses to oxidative stress. We also found that root and shoot growth of tcp9 mutants is less sensitive to exogenous hydrogen peroxide and that ROS accumulation in nematode infection sites in these mutants is reduced. Altogether, these observations demonstrate that TCP9 modulates the root system architectural plasticity to nematode infections via ROS-mediated processes. Our study further points at a novel regulatory mechanism contributing to the tolerance of plants to root-feeding nematodes by mitigating the impact of belowground biotic stresses.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Globodera pallida Genome Sequencing and Assembly, Globodera pallida Genome Sequencing and Assembly

This data was used for the creation of two high quality reference genomes., Background Potato cyst nematodes belong to the most harmful pathogens in potato, and durable management of these parasites largely depends on host-plant resistances. These resistances are pathotype specific. The current Globodera rostochiensis pathotype scheme that defines five pathotypes (Ro1 - Ro5) is both fundamentally and practically of limited value. Hence, resistant potato varieties are used worldwide in a poorly informed manner. Results We generated two novel reference genomes of G. rostochiensis inbred lines derived from a Ro1 and a Ro5 population. These genome sequences comprise 173 and 189 scaffolds respectively, marking a ≈ 24-fold reduction in fragmentation as compared to the current reference genome. We provide copy number variations for 19 effector families. Four dorsal gland effector families were investigated in more detail. SPRYSECs, known to be implicated in plant defence suppression, constitute by far the most diversified family studied herein with 60 and 99 variants in Ro1 and Ro5 distributed over 18 and 26 scaffolds. In contrast, CLEs, effectors involved in feeding site induction, show strong physical clustering. The 10 and 16 variants cluster on respectively 2 and 1 scaffolds. Given that pathotypes are defined by their effectoromes, we pinpoint the disparate nature of the contributing effector families in terms of sequence diversification and loss and gain of variants. Conclusions Two novel reference genomes allow for nearly complete inventories of effector diversification and physical organisation within and between pathotypes. Combined with insights we provide on effector family-specific diversification patterns, this constitutes a basis for an effectorome-based virulence scheme for this notorious pathogen.

Background: Potato cyst nematodes belong to the most harmful pathogens in potato, and durable management of these parasites largely depends on host-plant resistances. These resistances are pathotype specific. The current Globodera rostochiensis pathotype scheme that defines five pathotypes (Ro1 - Ro5) is both fundamentally and practically of limited value. Hence, resistant potato varieties are used worldwide in a poorly informed manner. Results: We generated two novel reference genomes of G. rostochiensis inbred lines derived from a Ro1 and a Ro5 population. These genome sequences comprise 173 and 189 scaffolds respectively, marking a ≈ 24-fold reduction in fragmentation as compared to the current reference genome. We provide copy number variations for 19 effector families. Four dorsal gland effector families were investigated in more detail. SPRYSECs, known to be implicated in plant defence suppression, constitute by far the most diversified family studied herein with 60 and 99 variants in Ro1 and Ro5 distributed over 18 and 26 scaffolds. In contrast, CLEs, effectors involved in feeding site induction, show strong physical clustering. The 10 and 16 variants cluster on respectively 2 and 1 scaffolds. Given that pathotypes are defined by their effectoromes, we pinpoint the disparate nature of the contributing effector families in terms of sequence diversification and loss and gain of variants. Conclusions: Two novel reference genomes allow for nearly complete inventories of effector diversification and physical organisation within and between pathotypes. Combined with insights we provide on effector family-specific diversification patterns, this constitutes a basis for an effectorome-based virulence scheme for this notorious pathogen.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

BackgroundRoot-knot nematodes transform vascular host cells into permanent feeding structures to withdraw nutrients from the host plant. Ecotypes of Arabidopsis thaliana can display large quantitative variation in susceptibility to the root-knot nematode Meloidogyne incognita, which is thought to be independent of dominant major resistance genes. However, in an earlier genome-wide association study of the interaction between Arabidopsis and M. incognita we identified a quantitative trait locus harboring homologs of dominant resistance genes but with minor effect on susceptibility to the M. incognita population tested.ResultsHere, we report on the characterization of two of these genes encoding the TIR-NB-LRR immune receptor DSC1 (DOMINANT SUPPRESSOR OF Camta 3 NUMBER 1) and the TIR-NB-LRR-WRKY-MAPx protein WRKY19 in nematode-infected Arabidopsis roots. Nematode infection studies and whole transcriptome analyses using the Arabidopsis mutants showed that DSC1 and WRKY19 co-regulate susceptibility of Arabidopsis to M. incognita.ConclusionGiven the head-to-head orientation of DSC1 and WRKY19 in the Arabidopsis genome our data suggests that both genes may function as a TIR-NB-LRR immune receptor pair. Unlike other TIR-NB-LRR pairs involved in dominant disease resistance in plants, DSC1 and WRKY19 most likely regulate basal levels of immunity to root-knot nematodes.

Systems genetics combines high-throughput genomic data with genetic analysis. In this chapter, we review and discuss application of systems genetics in the context of evolutionary studies, in which high-throughput molecular technologies are being combined with quantitative trait locus (QTL) analysis in segregating populations.The recent explosion of high-throughput data-measuring thousands of RNAs, proteins, and metabolites, using deep sequencing, mass spectrometry, chromatin, methyl-DNA immunoprecipitation, etc.-allows the dissection of causes of genetic variation underlying quantitative phenotypes of all types. To deal with the sheer amount of data, powerful statistical tools are needed to analyze multidimensional relationships and to extract valuable information and new modes and mechanisms of changes both within and between species. In the context of evolutionary computational biology, a well-designed experiment and the right population can help dissect complex traits likely to be under selection using proven statistical methods for associating phenotypic variation with chromosomal locations.Recent evolutionary expression QTL (eQTL) studies focus on gene expression adaptations, mapping the gene expression landscape, and, tentatively, define networks of transcripts and proteins that are jointly modulated sets of eQTL networks. Here, we discuss the possibility of introducing an evolutionary "prior" in the form of gene families displaying evidence of positive selection, and using that prior in the context of an eQTL experiment for elucidating host-pathogen protein-protein interactions.Here we review one exemplar evolutionairy eQTL experiment and discuss experimental design, choice of platforms, analysis methods, scope, and interpretation of results. In brief we highlight how eQTL are defined; how they are used to assemble interacting and causally connected networks of RNAs, proteins, and metabolites; and how some QTLs can be efficiently converted to reasonably w

Systems genetics combines high-throughput genomic data with genetic analysis. In this chapter, we review and discuss application of systems genetics in the context of evolutionary studies, in which high-throughput molecular technologies are being combined with quantitative trait locus (QTL) analysis in segregating populations.The recent explosion of high-throughput data-measuring thousands of RNAs, proteins, and metabolites, using deep sequencing, mass spectrometry, chromatin, methyl-DNA immunoprecipitation, etc.-allows the dissection of causes of genetic variation underlying quantitative phenotypes of all types. To deal with the sheer amount of data, powerful statistical tools are needed to analyze multidimensional relationships and to extract valuable information and new modes and mechanisms of changes both within and between species. In the context of evolutionary computational biology, a well-designed experiment and the right population can help dissect complex traits likely to be under selection using proven statistical methods for associating phenotypic variation with chromosomal locations.Recent evolutionary expression QTL (eQTL) studies focus on gene expression adaptations, mapping the gene expression landscape, and, tentatively, define networks of transcripts and proteins that are jointly modulated sets of eQTL networks. Here, we discuss the possibility of introducing an evolutionary "prior" in the form of gene families displaying evidence of positive selection, and using that prior in the context of an eQTL experiment for elucidating host-pathogen protein-protein interactions.Here we review one exemplar evolutionairy eQTL experiment and discuss experimental design, choice of platforms, analysis methods, scope, and interpretation of results. In brief we highlight how eQTL are defined; how they are used to assemble interacting and causally connected networks of RNAs, proteins, and metabolites; and how some QTLs can be efficiently converted to reasonably w

Root-knot nematodes transform vascular host cells into permanent feeding structures to selectively withdraw their nutrients from host plants during the course of several weeks. The susceptibility of host plants to root-knot nematode infections is thought to be a complex trait involving many genetic loci. However, genome-wide association (GWA) analysis has so far revealed only four quantitative trait loci (QTLs) linked to the reproductive success of the root-knot nematode Meloidogyne incognita in Arabidopsis thaliana, which suggests that the genetic architecture underlying host susceptibility could be much simpler than previously thought. Here, we report that, by using a relaxed stringency approach in a GWA analysis, we could identify 15 additional loci linked to quantitative variation in the reproductive success of M. incognita in Arabidopsis. To test the robustness of our analysis, we functionally characterized six genes located in a QTL with the lowest acceptable statistical support and smallest effect size. This led us to identify ETHYLENE RESPONSE FACTOR 6 (ERF6) as a novel susceptibility gene for M. incognita in Arabidopsis. ERF6 functions as a transcriptional activator and suppressor of genes in response to various abiotic stresses independent of ethylene signalling. However, whole-transcriptome analysis of nematode-infected roots of the Arabidopsis erf6-1 knockout mutant line showed that allelic variation at this locus may regulate the conversion of aminocyclopropane-1-carboxylate (ACC) into ethylene by altering the expression of 1-aminocyclopropane-1-carboxylate oxidase 3 (ACO3). Our data further suggest that tolerance to abiotic stress mediated by ERF6 forms a novel layer of control in the susceptibility of Arabidopsis to M. incognita.

Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.

Systems genetics combines high-throughput genomic data with genetic analysis. In this chapter, we review and discuss application of systems genetics in the context of evolutionary studies, in which high-throughput molecular technologies are being combined with quantitative trait locus (QTL) analysis in segregating populations. The recent explosion of high-throughput data—measuring thousands of RNAs, proteins, and metabolites, using deep sequencing, mass spectrometry, chromatin, methyl-DNA immunoprecipitation, etc.—allows the dissection of causes of genetic variation underlying quantitative phenotypes of all types. To deal with the sheer amount of data, powerful statistical tools are needed to analyze multidimensional relationships and to extract valuable information and new modes and mechanisms of changes both within and between species. In the context of evolutionary computational biology, a well-designed experiment and the right population can help dissect complex traits likely to be under selection using proven statistical methods for associating phenotypic variation with chromosomal locations. Recent evolutionary expression QTL (eQTL) studies focus on gene expression adaptations, mapping the gene expression landscape, and, tentatively, define networks of transcripts and proteins that are jointly modulated sets of eQTL networks. Here, we discuss the possibility of introducing an evolutionary “prior” in the form of gene families displaying evidence of positive selection, and using that prior in the context of an eQTL experiment for elucidating host-pathogen protein-protein interactions. Here we review one exemplar evolutionairy eQTL experiment and discuss experimental design, choice of platforms, analysis methods, scope, and interpretation of results. In brief we highlight how eQTL are defined; how they are used to assemble interacting and causally connected networks of RNAs, proteins, and metabolites; and how some QTLs can be efficiently converted to reasonably w

In this experiment we compare the effect of T-DNA insertion in DSC1 and WRKY19 during M. incognita infection. Where we compare the infected roots to uninfected roots at dpi 0 as the start of the infection and 7dpi, 7 days after inoculation. Both mutants are compared against Col-0 the wildtype plant.

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitatebinding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

Heligmosomoides polygyrus bakeri is a model parasitic hookworm used to study animal and human helminth diseases. During infection, the parasite releases excretory/secretory products that modulate the immune system of the host. The most abundant protein family in excretory/secretory products comprises the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (sperm-coating protein/ Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. There are >30 secreted Heligmosomoides polygyrus VAL proteins (HpVALs) and these proteins are characterised by having either one or two 15 kDa CAP (cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1) domains. The first known HpVAL structure, HpVAL-4, refined to 1.9 Å is reported. HpVAL-4 was produced as a homogeneously glycosylated protein in leaves of Nicotiana benthamiana infiltrated with recombinant plasmids, making this plant expression platform amenable for the production of biological products. The overall topology of HpVAL-4 is a three layered aba sandwich between a short N-terminal loop and a C-terminal cysteine rich extension. The C-terminal cysteine rich extension has two strands stabilized by two disulfide bonds and superposes well with the previously reported extension from the human hookworm Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2). The N-terminal loop is connected to alpha helix 2 via a disulfide bond previously observed in Na-ASP-2. HpVAL-4 has a central cavity that is more similar to the Nterminal CAP domain of the two CAP Na-ASP-1 from Necator americanus. Unlike Na-ASP-2, mammalian CRISP, and the C-terminal CAP domain of Na-ASP-1, the large central cavity of HpVAL-4 lacks the two histidines required to coordinate divalent cations. HpVAL-4 has both palmitate-binding and sterol-binding cavities and is able to complement the in vivo sterol export phenotype of yeast mutants lacking their endogenous CAP proteins. More studies are required to determine endogenous bin

Parallel adaptations enabling the use of plant cells as the primary food source have occurred multiple times in distinct nematode clades. The hallmark of all extant obligate and facultative plant-feeding nematodes is the presence of an oral stylet, which is required for penetration of plant cell walls, delivery of pharyngeal gland secretions into host cells and selective uptake of plant assimilates. Plant parasites from different clades, and even within a single clade, display a large diversity in feeding behaviours ranging from short feeding cycles on single cells to prolonged feeding on highly sophisticated host cell complexes. Despite these differences, feeding of nematodes frequently (but certainly not always) induces common responses in host cells (e.g. endopolyploidization and cellular hypertrophy). It is thought that these host cell responses are brought about by the interplay of effectors and other biological active compounds in stylet secretions of feeding nematodes, but this has only been studied for the most advanced sedentary plant parasites. In fact, these responses are thought to be fundamental for prolonged feeding of sedentary plant parasites on host cells. However, as we discuss in this review, some of these common plant responses to independent lineages of plant parasitic nematodes might also be generic reactions to cell stress and as such their onset may not require specific inputs from plant parasitic nematodes. Sedentary plant parasitic nematodes may utilize effectors and their ability to synthesize other biologically active compounds to tailor these common responses for prolonged feeding on host cells.

Susceptibility to the root-knot nematode Meloidogyne incognita in plants is thought to be a complex trait based on multiple genes involved in cell differentiation, growth and defence. Previous genetic analyses of susceptibility to M. incognita have mainly focused on segregating dominant resistance genes in crops. It is not known if plants harbour significant genetic variation in susceptibility to M. incognita independent of dominant resistance. To study the genetic architecture of susceptibility to M. incognita, we analysed nematode reproduction on a highly diverse set of 340 natural inbred lines of Arabidopsis thaliana with genome-wide association mapping. We observed a surprisingly large variation in nematode reproduction among these lines. Genome-wide association mapping revealed four quantitative trait loci (QTLs) located on chromosomes 1 and 5 of A. thaliana significantly associated with reproductive success of M. incognita, none of which harbours typical resistance gene homologues. Mutant analysis of three genes located in two QTLs showed that the transcription factor BRASSINAZOLE RESISTANT1 and an F-box family protein may function as (co-)regulators of susceptibility to M. incognita in Arabidopsis. Our data suggest that breeding for loss-of-susceptibility, based on allelic variants critically involved in nematode feeding, could be used to make crops more resilient to root-knot nematodes.

Aim: Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses. Methodology: Dendritic cells and macrophages were differentiated from mouse bone marrow and treated or depleted from GM-CSF prior to analyze their response to IL-10. Activity was assessed by measuring cytokine expression upon lipopolysaccharide stimulation, IL-10-induced signaling and downstream gene expression. Conclusion: This study describes that GM-CSF negatively regulates IL-10-mediated responses.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality.Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a key role in maintainingimmune homeostasis. IL-10-mediated responses are triggered upon binding to a heterodimericreceptor complex consisting of IL-10 receptor (IL-10R)1 and IL-10R2. Engagementof the IL-10R complex activates the intracellular kinases Jak1 and Tyk2, but the exact rolesof IL-10R2 and IL-10R2-associated signaling via Tyk2 remain unclear. To elucidate the contributionof IL-10R2 and its signaling to IL-10 activity, we re-evaluated IL-10-mediatedresponses on bone marrow-derived dendritic cells, macrophages and mast cells. By usingbone marrow from IL-10R-/- mice it was revealed that IL-10-mediated responses depend onboth IL-10R1 and IL-10R2 in all three cell types. On the contrary, bone marrow-derived cellsfrom Tyk2-/- mice showed similar responses to IL-10 as wild-type cells, indicating that signalingvia this IL-10R2-associated kinase only plays a limited role. Tyk2 was shown to controlthe amplitude of STAT3 activation and the up-regulation of downstream SOCS3 expression.SOCS3 up-regulation was found to be cell-type dependent and correlated with the lack ofearly suppression of LPS-induced TNF-α in dendritic cells. Further investigation of the IL-10R complex revealed that both the extracellular and intracellular domains of IL-10R2 influencethe conformation of IL-10R1 and that both domains were required for transducing IL-10 signals. This observation highlights a novel role for the intracellular domain of IL-10R2 inthe molecular mechanisms of IL-10R activation.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against a myriad of pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only few sequence changes have been shown to be sufficient to alter resistance specificities towards novel strains of a pathogen. However, little is known about the flexibility of NB-LRR genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato to the cyst nematode Globodera pallida and Potato virus X (PVX), respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) resulted in autoactivity, but lowering the protein levels restored its specific activation response including extreme resistance to PVX in potato shoots. The reciprocal construct (Rx1CN/Gpa2L) showed a loss-of-function phenotype, but exchange of the first 3 LRR repeats of Rx1 was sufficient to regain a wild type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either above or belowground. These data show the versatility of NB-LRR genes to generate resistances to unrelated pathogens with completely different lifestyles and routes of invasion.

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

Mushrooms are well known for their immunomodulating capacities. However, little is known about how mushroom-stimulated dendritic cells (DCs) affect T cells. Therefore, we investigated the effect of mushroom compounds derived from seven edible mushroom species on DCs, their fate in DCs, and the effect of the mushroom-stimulated DCs on T cells. Each mushroom species stimulated DCs in a different manner as was revealed from the DC’s cytokine response. Assessing DC maturation revealed that only one mushroom species, Agaricus subrufescens, induced complete DC maturation. The other six mushroom species upregulated MHC-II and CD86 expression, but did not significantly affect the expression of CD40 and CD11c. Nevertheless, mushroom compounds of all investigated mushroom species are endocytosed by DCs. Endocytosis is most likely mediated by C-type lectin receptors (CLRs) because CLR binding is Ca2+ dependent, and EGTA reduces TNF-α secretion with more than 90%. Laminarin partly inhibited TNF-α secretion indicating that the CLR dectin-1, among other CLRs, is involved in binding mushroom compounds. Stimulated DCs were shown to stimulate T cells; however, physical contact of DCs and T cells is not required. Because CLRs seem to play a prominent role in DC stimulation, mushrooms may function as a carbohydrate containing adjuvant to be used in conjunction with anti-fungal vaccines.

Intensification of conventional agriculture has resulted in a decline of soil ecosystem functioning. Organic agriculture intends to manage soil biota in a manner that is more geared towards adequate cycling of nutrients with minimal losses. Ecological interpretation of agricultural practices-induced shifts in primary decomposers, bacteria and fungi, is non-trivial due to their enormous biodiversity. Bacterivorous and fungivorous nematodes feed selectively on these microorganisms, and we intended to test whether farming system effects are mirrored in compositional changes in nematode communities. Therefore, we analysed the impact of three farming systems, conventional (ConMin), integrated (ConSlu) and organic (Organic), on nematode communities in the southeastern part of The Netherlands on a sandy soil with 3–5% organic matter. Effects of each farming system were assessed for four different crops (barley, maize, pea or potato) by a series of taxon-specific quantitative PCRs (qPCR). Changes in community structure analysed by nonmetric multidimensional scaling (NMDS) showed that organic farming resulted in specific shifts in nematode community composition exceeding crop-related assemblage shifts. Three out of thirteen quantified nematode taxa showed significant farming system effects. Strongest effects were observed for the (putative) bacterivore Prismatolaimus, which was relatively common in Organic fields and nearly absent in ConMin and ConSlu fields. A reverse effect was observed for Pristionchus; this necromenic bacterivore and facultative predator made up about 21% and 7% of the total nematode community in respectively ConMin and ConSlu fields, whereas it was nearly absent from Organic fields. The observed farming system effects suggest that specific nematode taxa might be indicative for the impact of farming practices on soil biota.

Intensification of conventional agriculture has resulted in a decline of soil ecosystem functioning. Organic agriculture intends to manage soil biota in a manner that is more geared towards adequate cycling of nutrients with minimal losses. Ecological interpretation of agricultural practices-induced shifts in primary decomposers, bacteria and fungi, is non-trivial due to their enormous biodiversity. Bacterivorous and fungivorous nematodes feed selectively on these microorganisms, and we intended to test whether farming system effects are mirrored in compositional changes in nematode communities. Therefore, we analysed the impact of three farming systems, conventional (ConMin), integrated (ConSlu) and organic (Organic), on nematode communities in the southeastern part of The Netherlands on a sandy soil with 3–5% organic matter. Effects of each farming system were assessed for four different crops (barley, maize, pea or potato) by a series of taxon-specific quantitative PCRs (qPCR). Changes in community structure analysed by nonmetric multidimensional scaling (NMDS) showed that organic farming resulted in specific shifts in nematode community composition exceeding crop-related assemblage shifts. Three out of thirteen quantified nematode taxa showed significant farming system effects. Strongest effects were observed for the (putative) bacterivore Prismatolaimus, which was relatively common in Organic fields and nearly absent in ConMin and ConSlu fields. A reverse effect was observed for Pristionchus; this necromenic bacterivore and facultative predator made up about 21% and 7% of the total nematode community in respectively ConMin and ConSlu fields, whereas it was nearly absent from Organic fields. The observed farming system effects suggest that specific nematode taxa might be indicative for the impact of farming practices on soil biota.

Background: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. Results: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. Conclusions: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly reflected in this thesis. Bioinformaticians often contribute crucial insights and novelty to scientific research because they are central to data analysis and contribute concrete algorithms and software solutions. In addition, bioinformaticians have an important role to play when it comes to organising data and software and making it accessible to others. In this thesis, in addition to contributing to biological questions, I discuss issues around accessing and sharing data, with the challenges of handling large data, input/output (IO) bottlenecks and effective use of multi-core computations. By creating software solutions together with molecular biologists, I contributed and published insights in biological processes in nematodes and plants. I published software solutions that made it easier for others to analyse data, which impacts the wider research community. I created solutions that made it easier for others to publish software solutions by themselves. The introduction of computing and the internet makes it possible to share ideas and computational methods. I am convinced it is a good idea to publish software solutions as `free and open source' software (FOSS) in the public domain so that we can continue to build on the work of others. Chapter 2 presents a computational method for identifying gene families in a sequenced genome that may be involved in pathogenicity, i.e., those genes that code for proteins that interact with molecules of an infected host. Such nematode proteins are known to contain highly variable DNA sections that code for the biochemical properties of an interaction site. By applying phylogenetic analysis through maximum likelihood (PAML) and comparison

Parasitic worms threaten human, animal and plant health by infecting people, livestock and crops worldwide. Animals and plants share an anciently evolved innate immune system. Parasites modulate this immune system by secreting proteins to maintain their parasitic lifestyle. This thesis describes how venom-allergen-like proteins (VAPs) that both animal- and plant-parasitic nematodes release into their hosts, modulate host innate immunity. On the one hand we found that one particular secreted VAP from the potato cyst nematode can activate host defenses in tomato plants, opening an opportunity for plant breeders to generate novel nematode-resistant cultivars. We showed that plants make more efficiently use of their limited repertoire of immune receptors by guarding common virulence targets of multiple unrelated plant pathogens. While on the other hand, we describe how VAPs may be used by parasites to suppress the host defense responses mediated by extracellular immune receptors. In short, this fundamental study contributes to our understanding of the molecular basis of persistent infections by parasitic nematodes in plants and in animals.

Nematodes are probably the most abundant Metazoans on our planet. They are present in densities of millions of individuals per square meter in soil and sediments. However, these inconspicuous animals are hardly known to the general public as most individuals are colorless and smaller than 1 mm. If people are aware of nematodes, it is because of the damage they inflict on humans and animals such as elephantiasis (Wucheria bancrofti) and ascariasis (in humans Ascaris lubricoides; in pigs Ascaris suum), or on plants such as potato (potato cyst nematode) and tomato (root-knot nematodes). To a far lesser extent it is known that majority of nematodes are key players in the soil food web, and as such they can be used as indicators for the biological condition of the environment they live in. This PhD thesis focuses on terrestrial nematodes belonging to four orders: Dorylaimida, Mononchida, Aphelenchida and Tylenchida, which represent animals of ecological and economical relevance. Members of families belonging to the first two are highly sensitive to the environmental disturbances, and are informative as biological indicators. The order Aphelenchida harbors numerous facultative plant-parasitic species. In the absence of a host plant, most of them are able to feed on fungi as an alternative food source. This is in contrast to the distal representatives of the order Tylenchida that are invariably obligate parasites of higher plants. Stress-sensitive nematode orders Dorylaimida and Mononchida have a high potential for soil health assessment. The SSU rDNA-based analysis of these two orders resulted in two highly distinct phylogenies. Relationships among the Mononchida, an order dominated by carnivorous nematodes, were to some extent in accordance with the classical nematode systematics. It is noted that the families Mylonchulidae, Mononchidae and Anatonchidae are not monophyletic. Nevertheless, it was possible to design family-specific primers for rDNA-based molecular detectio

Plant-parasitic cyst nematodes are major agricultural pests worldwide. These obligate endoparasites invade the roots of host plants where they transform cells near the vascular cylinder into a permanent feeding site. Plants possess a multilayered innate immune system consisting of different types of extracellular and intracellular immune receptors. These enable detection of most invading nematodes and initiate immune responses that result in resistance. Many plant pathogens use effectors to overcome resistance. Here, modulation of plant innate immunity by plant-parasitic cyst nematodes was investigated. Extracellular immune receptor signaling and hormone-mediated signaling pathways were found to contain infection of susceptible Arabidopsis thalianawith Heterodera schachtii. A large family of effectors was identified in Globodera rostochiensis. One of these so-called SPRYSECs interacted with a novel CC-NB-LRR type resistance protein of a susceptible tomato without inducing resistance responses. Instead, the effector was found to suppress defense-related programmed cell death and resistance mediated by several CC-NB-LRR type resistance proteins. In addition, a secreted antimicrobial peptide was identified in G. rostochiensis. Plant-parasitic cyst nematodes thus most likely secrete effectors that protect against plant immune responses and secondary infections. The current evidence for the existence of immune modulating effectors is reviewed and directions for further research are given.

Biogem provides a software development environment for the Ruby programming language, which encourages community-based software development for bioinformatics while lowering the barrier to entry and encouraging best practices. Biogem, with its targeted modular and decentralized approach, software generator, tools and tight web integration, is an improved general model for scaling up collaborative open source software development in bioinformatics.

Sedentary plant parasitic nematodes have evolved sophisticated strategies that allow them to transform host cells in the roots of host plants into feeding structures. These complex structures enable the nematodes to complete their life cycle inside a single host plant. Feeding structure initiation and maintenance are thought to be determined by the concerted action of effectors produced by the esophageal glands of the nematodes. However, the molecular mechanisms underlying the transformation of host cells into feeding structures and the role of the effectors in this process are poorly understood. For example, it is generally thought that virulent nematodes also use effectors to protect their complex feeding structures from plant innate immune responses. However, nematode effectors suppressing plant innate immunity have not been identified to date. The host ranges of sedentary plant parasitic nematodes can vary from very wide (e.g. root-knot nematodes) to relatively narrow, limited to single plant families (e.g. cyst nematodes). Potato cyst nematodes (PCN) are able to parasitize only Solanaceous plants. Every year, they cause substantial yield losses in potato production areas. PCN are very difficult to control by the use of cultivation methods or the application of pesticides alone. The introduction of novel nematode resistant potato cultivars to the market is therefore of great importance for potato growers all over the world. Resistances, however, can be overcome by the emergence of virulent nematode populations. The aim of this thesis is to study incompatible interactions between the potato cyst nematode Globodera rostochiensis and potato (Solanum tuberosum), by analyzing resistance loci that make the plant immune to potato cyst nematodes as well as nematode effectors that suppress plant innate immunity. This thesis begins with an extensive review of the literature on molecular and cellular aspects of plant resistance to sedentary endoparasitic nematodes, includi

Gpa2 recognition specificity Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety of free living nematodes feeding on bacteria can be found as well as species that parasitize insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell complex or so-called syncytium, on which the immobilized nematode fully depends for its development and reproduction. Due to the sophisticated feeding manner and ability to survive for a long time in the absence of a host plant, the best way to control these soil-born pathogens is the exploitation host resistance. Natural resistance to nematodes is based on single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X (Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in 88% identical at the amino acid level. The vast majority of the differences between Gpa2 and Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we have shown that the LRR domain is essential for the recognition specificities of Gpa2 and Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR were replaced by the corresponding segments from Rx1. These constructs allowed us to narrow down the region required for nematode recognition to a stretch of residues between 808 and 912 amino acid residues in Gpa2, including 10 amino acids that di