Geert Smant

Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains.

The resistance protein Rx1 exists in cytoplasmic and nuclear pools in the cell. Both subcellular pools are necessary for full PVX resistance, and the cytoplasmic compartment could be linked to PVX recognition. A functional phosphate binding loop and the presence of SGT1 are required to sustain the nuclear pool. Functional domains of Rx1 were shown to have opposing roles in Rx1 localization. The Rx1 protein, as many resistance proteins of the nucleotide binding–leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.

The Activation and Suppression of Plant Innate Immunity by Parasitic Nematodes

Plant-parasitic nematodes engage in prolonged and intimate relationships with their host plants, often involving complex alterations in host cell morphology and function. It is puzzling how nematodes can achieve this, seemingly without activating the innate immune system of their hosts. Secretions released by infective juvenile nematodes are thought to be crucial for host invasion, for nematode migration inside plants, and for feeding on host cells. In the past, much of the research focused on the manipulation of developmental pathways in host plants by plant-parasitic nematodes. However, recent findings demonstrate that plant-parasitic nematodes also deliver effectors into the apoplast and cytoplasm of host cells to suppress plant defense responses. In this review, we describe the current insights in the molecular and cellular mechanisms underlying the activation and suppression of host innate immunity by plant-parasitic nematodes along seven critical evolutionary and developmental transitions in plant parasitism.

The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

BackgroundThe potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.ResultsThe recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.ConclusionThis is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

A rapid and massive gene expression shift marking adolescent transition in C. elegans

Organismal development is the most dynamic period of the life cycle, yet we have only a rough understanding of the dynamics of gene expression during adolescent transition. Here we show that adolescence in Caenorhabditis elegans is characterized by a spectacular expression shift of conserved and highly polymorphic genes. Using a high resolution time series we found that in adolescent worms over 10,000 genes changed their expression. These genes were clustered according to their expression patterns. One cluster involved in chromatin remodelling showed a brief up-regulation around 50u2005h post-hatch. At the same time a spectacular shift in expression was observed. Sequence comparisons for this cluster across many genotypes revealed diversifying selection. Strongly up-regulated genes showed signs of purifying selection in non-coding regions, indicating that adolescence-active genes are constrained on their regulatory properties. Our findings improve our understanding of adolescent transition and help to eliminate experimental artefacts due to incorrect developmental timing.

Fast probabilistic file fingerprinting for big data

BackgroundBiological data acquisition is raising new challenges, both in data analysis and handling. Not only is it proving hard to analyze the data at the rate it is generated today, but simply reading and transferring data files can be prohibitively slow due to their size. This primarily concerns logistics within and between data centers, but is also important for workstation users in the analysis phase. Common usage patterns, such as comparing and transferring files, are proving computationally expensive and are tying down shared resources.ResultsWe present an efficient method for calculating file uniqueness for large scientific data files, that takes less computational effort than existing techniques. This method, called Probabilistic Fast File Fingerprinting (PFFF), exploits the variation present in biological data and computes file fingerprints by sampling randomly from the file instead of reading it in full. Consequently, it has a flat performance characteristic, correlated with data variation rather than file size. We demonstrate that probabilistic fingerprinting can be as reliable as existing hashing techniques, with provably negligible risk of collisions. We measure the performance of the algorithm on a number of data storage and access technologies, identifying its strengths as well as limitations.ConclusionsProbabilistic fingerprinting may significantly reduce the use of computational resources when comparing very large files. Utilisation of probabilistic fingerprinting techniques can increase the speed of common file-related workflows, both in the data center and for workbench analysis. The implementation of the algorithm is available as an open-source tool named pfff, as a command-line tool as well as a C library. The tool can be downloaded from http://biit.cs.ut.ee/pfff.