Vivian C. Blok

Efficient Virus-Induced Gene Silencing in Roots Using a Modified Tobacco Rattle Virus Vector

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and β-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the proteins interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.

Characterization of a chorismate mutase from the potato cyst nematode Globodera pallida

SUMMARY Some plant endoparasitic nematodes are biotrophic and induce remarkable changes in their hosts in order to ensure a continuous supply of food. Proteins secreted from oesophageal gland cells have been implicated in this pathogenic process. A potentially secreted chorismate mutase has been isolated from the potato cyst nematode Globodera pallida. The gene encoding this protein is expressed in the subventral oesophageal gland cells of the nematode, and the mRNA derived from this gene is only present in the early parasitic stages. Sequence analysis of this gene shows that, like other genes involved in the host-parasite interaction of plant parasitic nematodes, it is likely to have been acquired by horizontal gene transfer from bacteria. The presence of a signal peptide in the deduced amino acid sequence of the G. pallida chorismate mutase and its expression in the subventral oesophageal gland cells suggest that it is secreted from the nematode, pointing to a role for the protein in the host-parasite interaction. The shikimate pathway, of which chorismate mutase is normally a part, is not found in animals but is present in plants and bacteria. In plants it gives rise to a variety of compounds which are important in amino acid synthesis and defence signalling pathways, as well as auxins, which have been implicated in the early development of nematode feeding sites. The potential roles of a nematode chorismate mutase are discussed.

Analysis of genes expressed in second stage juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida using the expressed sequence tag approach.

Expressed sequence tag (EST) projects offer a rapid route to the discovery of novel genes. Genes expressed in a wide range of parasitic nematodes of medical or veterinary importance have been investigated using EST analysis but these techniques have not yet been applied to plant parasitic nematodes. We describe a small scale EST project using cDNA libraries made from the two species of potato cyst nematode, Globodera rostochiensis and G. pallida, and assess the utility of this approach to identify mRNAs encoding abundantly expressed secreted proteins and other proteins present in the nematode at the onset of parasitism. Approximately 1000 sequences were obtained from G. rostochiensis and 100 from G. pallida. A variety of genes was characterised and approximately 11% of the cDNAs sequenced were apparently PCN specific. Secreted proteins identified included a novel PCN homologue of chorismate mutase, a cDNA recently cloned from the gland cells of Meloidogyne javanica. The results obtained justify a much larger scale application of this technology to these parasites. Utilisation de LExpressed Sequence Tag pour lanalyse de genes sexprimant chez les juveniles de deuxieme stade des nematodes a kyste de la pomme de terre Globodera rostochiensis et G. pallida - lExpressed Sequence Tag (EST) ouvre une voie rapide vers la decouverte de nouveaux genes. Des genes sexprimant chez un large eventail de nematodes parasites dimportance medicale ou veterinaire ont ete etudies par analyse EST alors que cette technique na pas encore ete appliquee aux nematodes phytoparasites. Nous decrivons ici un projet EST a petite echelle utilisant les banques dADNc constituees a partir de deux especes de nematodes a kyste de la pomme de terre (PCN), Globodera rostochiensis et G. pallida et nous evaluons lutilite de cette approche pour identifier les ARNs codant les proteines abondamment secretees - et les autres proteines - presentes chez les nematodes lors de lattaque parasitaire. Mille sequences environ ont ete obtenues chez G. rostochiensis et 100 chez G. pallida. Des genes varies ont ete caracterises et environ 11% des ADNc sequences sont apparemment specifiques des PCN. Parmi les proteines secretees identifiees figurent un nouvel homologue PCN de la chorismate mutase ainsi quun ADNc recemment clone a partir de cellules glandulaires de Meloidogyne javanica. Les resultats ainsi obtenus justifient une utilisation a plus grande echelle de lEST pour letude de ces parasites.

Occurrence of resistance-breaking populations of root-knot nematodes on tomato in Greece.

Nine populations of Meloidogyne spp. from Greece have been identified as M. javanica or M. incognita using either isozyme phenotypes or the sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) technique. Virulence against the Mi resistance gene was assayed by pot experiments in controlled conditions and revealed the ability of five populations of M.javanica and one population of M. incognita to reproduce on tomato cultivars containing that gene. A resistance-breaking population of M. incognita is reported for the first time in the country; the M. javanica populations constitute new records for the Greek mainland.

The Mitochondrial Subgenomes of the Nematode Globodera pallida Are Mosaics: Evidence of Recombination in an Animal Mitochondrial Genome

We sequenced four mitochondrial subgenomes from the potato cyst nematode Globodera pallida, previously characterized as one of the few animals to have a multipartite mitochondrial genome. The sequence data indicate that three of these subgenomic mitochondrial circles are mosaics, comprising long, multigenic fragments derived from fragments of the other circles. This pattern is consistent with the operation of intermitochondrial recombination, a process generally considered absent in animal mitochondria. We also report that many of the duplicated genes contain deleterious mutations, ones likely to render the gene nonfunctional; gene conversion does not appear to be homogenizing the different gene copies. The proposed nonfunctional copies are clustered on particular circles, whereas copies that are likely to code functional gene products are clustered on others.

Parasitism genes and host range disparities in biotrophic nematodes: the conundrum of polyphagy versus specialisation

This essay considers biotrophic cyst and root‐knot nematodes in relation to their biology, host–parasite interactions and molecular genetics. These nematodes have to face the biological consequences of the physical constraints imposed by the soil environment in which they live while their hosts inhabit both above and below ground environments. The two groups of nematodes appear to have adopted radically different solutions to these problems with the result that one group is a host specialist and reproduces sexually while the other has an enormous host range and reproduces by mitotic parthenogenesis. We consider what is known about the modes of parasitism used by these nematodes and how it relates to their host range, including the surprising finding that parasitism genes in both nematode groups have been recruited from bacteria. The nuclear and mitochondrial genomes of these two nematode groups are very different and we consider how these findings relate to the biology of the organisms. BioEssays 30:249–259, 2008.

SXP/RAL-2 proteins of the potato cyst nematode Globodera rostochiensis: secreted proteins of the hypodermis and amphids

A family of secreted proteins (the SXP/ RAL-2 proteins) has been identified in animal parasitic nematodes and Caenorhabditis elegans . In this paper, we describe two full length cDNA sequences from the potato cyst nematode Globodera rostochiensis which could encode proteins similar to SXP/ RAL-2 proteins. We show that although both genes are expressed in second stage juveniles and in sedentary females of G. rostochiensis , they have remarkably different spatial expression patterns. The mRNA derived from one of the genes ( gr-sxp-1 ) was present in the hypodermis, as has been described previously for one of these genes in an animal parasitic nematode. However, expression of the mRNA from the other gene was restricted to the gland cells surrounding the amphidial sense organs (the sheath cells). In addition to providing the first description of SXP proteins from a plant parasitic nematode, this work provides the first detailed characterisation of a protein secreted from the amphids of a plant parasite. Les proteines SXP/ RAL-2 du nematode a kyste de la pomme de terre Globodera rostochiensis : les proteines secretees par lhypoderme et les amphides - Une famille de proteines secretees - les proteines SXP/ RAL-2 - avait deja ete identifiee chez les nematodes zooparasites et chez Caenorhabditis elegans . Dans cet article nous decrivons deux sequences en longueur totale de cADN provenant du nematode a kyste de la pomme de terre Globodera rostochiensis , sequences qui pourraient coder des proteines similaires aux proteines SXP/ RAL-2 . Nous montrons que, bien que lun et lautre gene sexpriment chez les juveniles de deuxieme stade et chez les femelles sedentaires de Globodera rostochiensis , ils possedent des profils dexpression spatiale remarquablement differents. Le rARN derive a partir dun de ces genes ( gr-sxp-1 ) etait present dants lhypoderme, comme cela avait deja ete decrit pour lun de ces genes chez un nematode zooparasite. Cependant, lexpression du mRNA de lautre gene etait limitee aux cellules glandulaires entourant les organes sensoriels amphidiens (les cellules de la gaine). Offrant la premiere description de proteines SXP provenant de nematodes phytopathogenes, le present travail fournit de plus la premiere caracterisation detaillee dune proteine secretee par les amphides dun nematode parasite de plantes.

Identification of Globodera rostochiensis and G. pallida in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpats’ka oblast’), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.

Segregation and recombination of a multipartite mitochondrial DNA in populations of the potato cyst nematode globodera pallida

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods.