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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 Gpa2 CC-NB-LRR, which confers 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 defence.A N. benthamiana homolog of the Glycine-Rich RNA-Binding Protein 7 (NbGRP7) protein was prioritized as a novel 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 compromises its role in Rx1-mediated defence. Strikingly, ectopic NbGRP7 expression impacts the steady-state levels of Rx1, which relies on an intact RNA recognition motif.Combined, our findings illustrate that NbGRP7 is a novel pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 functioning at a post-transcriptional level.

The Gpa2 and Rx1 intracellular immune receptors are canonical CC-NB-LRR proteins belonging to the same R gene cluster in potato. Despite sharing high sequence homology, they have evolved to provide defence against unrelated pathogens. Gpa2 detects Gp-RBP-1 effectors secreted by the potato cyst nematode Globodera pallida whereas Rx1 recognizes the viral coat protein (CP) of Potato Virus X (PVX). How Gpa2 and Rx1 perceive their matching effectors remains unknown. Using a combination of in planta Co-Immunoprecipitation and cellular imaging, we show that both Gp-RBP-1 and PVX-CP physically interact with RanGAP2 and RanGAP1 in the cytoplasm of plant cells. Interestingly, this was also demonstrated for the eliciting variants of Gp-RBP-1 and PVX-CP indicating a role for RanGAP1 and RanGAP2 in pathogenicity independent from Gpa2 and Rx1 recognition. Indeed, knocking down both RanGAP homologs reduce cyst nematode and PVX infection. These findings show that RanGAP1/2 act as common host targets of evolutionary distinct effectors from two plant pathogens with different lifestyles. The involvement of RanGAP1/2 to pathogen virulence is a novel role not yet reported for these key host cell components and as such, their possible role in cyst nematode parasitism and viral pathogenicity are discussed. Moreover, from these findings a model emerges for their possible role as co-factor in pathogen recognition by the potato immune receptors Gpa2/Rx1.

Soil-borne cyst nematodes are obligatory sedentary parasites that cause severe losses to cultivation of major crops such as potato and soybean. Cyst nematodes establish specialised permanent feeding sites within the roots of their host by manipulating plant morphology and physiology through secreted effectors. Here we identified host targets of effector GpRbp-1 and studied their roles in plant-nematode interactions. GpRbp-1 was found to interact in yeast and in planta with the potato and Arabidopsis homologues of Siz/PIAS-type E3 SUMO ligase SIZ1. Our results show that a pathogen effector targets the master regulator SIZ1 in plant cells, which has not been demonstrated earlier to our knowledge. The interaction of GpRbp-1 and SIZ1 localizes to the plant nucleus, suggesting that the nuclear functions of SIZ1 as regulator of plant immunity and physiology may be modulated by GpRbp-1. Furthermore, nematode infection assays and transcriptomic profiling indicate that SIZ1 is required for susceptibility to cyst nematodes. So, these data indicate that E3 SUMO ligases may play an important role in plant-nematode interactions. Based on the prediction of SUMO acceptor and interaction sites in GpRbp-1, a model is proposed in which the effector may recruit SIZ1 to be SUMOylated for full functionality in host cells.

SE-vdA is supported by BBSRC grant BB/M014207/1. Sequencing was funded by BBSRC grant BB/F000642/1 to the University of Leeds and grant BB/F00334X/1 to the Wellcome Trust Sanger Institute). DRL was supported by a fellowship from The James Hutton Institute and the School of Biological Sciences, University of Edinburgh. GK was supported by a BBSRC PhD studentship. The James Hutton Institute receives funding from the Scottish Government. JAC and NEH are supported by the Wellcome Trust through its core funding of the Wellcome Trust Sanger Institute (grant 098051). This work was also supported by funding from the Canadian Safety and Security Program, project number CRTI09_462RD. 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. Publisher PDF

Sedentary plant-parasitic nematodes engage in a long-lasting and intimate relationship with their host plant. This interaction starts in the soil when freshly hatched infective juveniles are attracted to specific parts of a host plant root system. Little is known of what determines the attractiveness of host plant roots, but a mix of biochemical and structural cues from specific host tissues in the roots likely triggers invasive behavior in nematodes. Similarly to nematodes, phloem-feeding aphids and other piercing-sucking (hemipteran) insects must establish close associations with their host plants in order to modulate plant cellular processes to promote feeding and reproduction. This focus issue on molecular interactions between plants and different representatives of the animal kingdom sheds light on recent developments and offers a platform for exciting new data in this field. We are grateful to all the authors for their diverse contributions and for making this focus issue possible

Many plant and animal immune receptors have a modular NB-LRR architecture in which a nucleotide-binding switch domain (NB-ARC) is tethered to a leucine-rich repeat sensor domain (LRR). The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato Virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and the N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (position 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation and the complementary charged regions are predicted to mediate a fast re-association enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB and N-terminal half of the LRR form a clamp, which regulates the dissociation and re-association of the switch and sensor domains in NB-LRR proteins.

Genetical genomics combines acquired high-throughput genomic data with genetic analysis. In this chapter, we discuss the application of genetical genomics for evolutionary studies, where new high-throughput molecular technologies are combined with mapping quantitative trait loci (QTL) on the genome in segregating populations. The recent explosion of high-throughput data—measuring thousands of proteins and metabolites, deep sequencing, chromatin, and methyl-DNA immunoprecipitation—allows the study of the genetic variation underlying quantitative phenotypes, together termed xQTL. At the same time, mining information is not getting easier. To deal with the sheer amount of information, powerful statistical tools are needed to analyze multidimensional relationships. In the context of evolutionary computational biology, a well-designed experiment may help dissect a complex evolutionary trait using proven statistical methods for associating phenotypical variation with genomic locations. Evolutionary expression QTL (eQTL) studies of the last years focus on gene expression adaptations, mapping the gene expression landscape, and, tentatively, 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 in the context of an eQTL experiment for elucidating host–pathogen protein–protein interactions. Through the example of an experimental design, we discuss the choice of xQTL platform, analysis methods, and scope of results. The resulting eQTL can be matched, resulting in putative interacting genes and their regulators. In addition, a prior may help distinguish QTL causality from reactivity, or independence of traits, by creating QTL networks.

Aan Wageningen University zijn digitale lesmodules ontwikkeld op het gebied van plantenziektekunde en plantenveredeling. In 2005 is hiermee een begin gemaakt, met subsidies van de KNPV, samen met de stichting Willie Commelin Scholten voor de fytopathologie. Dit project laat zien dat relatief kleine initiatieven de aanjager kunnen zijn voor veel grotere projecten, die uiteindelijk ten goede komen aan de hele sector.

Wortelknobbelaaltjes (Meloidogyne spp.) behoren tot de meest geavanceerde parasieten onder de ziekteverwekkers van planten. Ze onderscheiden zich van veel obligaat biotrofe pathogenen door hun brede waardplantenreeks. Eenmaal gevestigd in het wortelstelsel onttrekken ze vervolgens gedurende enkele weken voedingsstoffen aan de plant via speciaal daarvoor aangelegde voedingsstructuren - de reuzecellen. Ze kunnen zich al naar gelang de omstandigheden dat vereisen, op allerlei manieren voortplanten.De laatste jaren zijn door de toepassing van moleculaire technieken een aantal bijzondere ontdekkingen gedaan die betrekking hebben op deze aanpassingen. Ingegaan wordt op de ontwikkelingen met nadruk op de genen, die betrokken zijn bij de parasitaire levensstijl van de nematode

Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity1. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls2, 3, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.

Durable parasitic relationships are fascinating and scientifically interesting as they illustrate the maximal impact of co-evolution. Often these parasites have evolved the ability to recognize individual compounds unintentionally produced by their hosts. These signals will then trigger the release of an array of pathogenicity factors from the parasites. Comprehension of the molecular events underlying the resulting intimate reactions will lead to a better understanding of fundamental biological processes as well as the development of new approaches to elegantly and specifically control harmful pests

We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain. The protein product GT-ENG-1 contains an additional 95 amino acid carboxy terminal sequence with strong similarity to type II cellulose binding domains. Riboprobes and polyclonal antibodies raised to recombinant cyst nematode EGases were used to follow expression patterns of EGase transcripts and proteins throughout the nematode life cycle. EGase transcripts and proteins were specifically detected within the subventral esophageal gland cells of G. tabacum second-stage juveniles (J2) within eggs prior to hatching, in preparasitic J2, and in parasitic J2 that had invaded tobacco roots. EGase transcripts and proteins were not detected in G. tabacum after the molt to the sedentary J3, J4, and adult female life stages. Interestingly, EGase transcription and translation resumed in the subventral esophageal glands of late J4 males. It is hypothesized that secreted EGases play a major role to facilitate intracellular migration of G. tabacum within tobacco roots.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

This thesis describes the identification ofβ-1,4-endoglucanases (cellulases) in stylet secretions of the two cyst nematodes species, Globodera rostochiensis and Heterodera glycines . A novel method was developed to raise monoclonal antibodies that were directed to subventral oesophageal gland secretions. These monoclonal antibodies were used to characterise and to immunopurify two secretory proteins. Partial sequence data from these proteins enabled the cloning of two homologous genes from each of the two cyst nematode species.The predicted amino acid sequences revealed a high similarity with bacterial cellulases, whereas no homology was found with eukaryotic cellulases. Evidence is provided for the endogenous origin the nematode cellulases that may have been acquired from bacteria by horizontal gene transfer.

Après immunisation de souris avec différents antigènes de #Globodera rostochiensis$, les anticorps monoclonaux (MAbs) ont été testés par microscopie en fluorescence pour leur réaction avec des structures particulières de juvéniles de deuxième stade (J2) de #G. rostochiensis$. Il a été obtenu des MAbs qui se lient avec les glandes oesophagiennes subventrales, les filaments musculaires de la paroi du corps, le primordium génital, la lumière intestinale, les noyaux des cellules et la surface de la cuticule. Les MAbs des glandes subventrales se lient également aux glandes subventrales des J2 de #G. pallida$ et #G. tabacum$, mais non à celles des J2 d'#Heterodera glycines$, #H. schachtii$, #Meloidogyne hapla$ ou #M. incognita$. Des MAbs des glandes subventrales réagissent avec un épitope hydrosoluble des extraits protéiques de J2 de #G. rostochiensis$. Les MAbs des filaments musculaires de la paroi du corps se lient également à la musculature de la paroi du corps des J2 de #G. pallida$, #H. schachtii$, #M. hapla$ et #M. incognita$. Par immunotransfert de J2 de #G. rostochiensis$, ces MAbs réagissent avec deux protéines de 39 et plus de 106 kDa, respectivement. (Résumé d'auteur)

The invention provides peptides having cellulase activity and exhibiting at least 40mino acid identity in the primary structure with the amino acid sequence shown in one of SEQ ID NO.'s 1, 2, 3 and 4 or comprising a series of at least 7 contiguous amino acids of the amino acid sequence shown in one of SEQ ID NO.'s 1, 2, 3 and 4. Said peptide corresponds to a nematodal peptide especially of a sedentary nematode (such as a cyst nematode or root-knot nematode) or part thereof. Antibodies against such peptides are valuable crop-protecting agents. Furthermore provided are nucleotide sequences encoding these peptides and expression systems comprising at least such a nucleotide sequence. The enzymic peptides are useful in the protection of plants against parasitic nematodes, and in the food and beverage industry, in paper or clothing industry or in waste treatment

Item does not contain fulltext Brief Communications p.

The yellow potato cyst nematode Globodera rostochiensis is a devastating plant pathogen of global economic importance, classified into pathotypes of different plant resistance-breaking phenotypes. G. rostochiensis secretes effectors, some of which were acquired by horizontal gene transfer (HGT), from pharyngeal glands into the host to manipulate host processes and promote parasitism. We generated a high-quality genome assembly for G. rostochiensis pathotype Ro1 and identified putative effectors and HGT events, mapped gene expression through the life cycle focusing on key parasitic transitions, and sequenced the genomes of eight populations including three additional pathotypes.