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Two homologues of map-1, which encodes a putative avirulence factor, were found in a single egg mass line of the root-knot nematode Meloidogyne javanica that was virulent to the Mi-resistant gene. The main difference between the two encoded proteins of these homologues, MJ-MAP-1 and MJ-MAP-2, was the position of a 13 amino acid repeat region. Genes encoding two related but novel proteins, MJAP-1 and MJAP-2 (290 and 283 amino acids, respectively, including a potential signal secretion peptide), were also isolated from M. javanica. They have high similarity to MAP at the C-terminus. MJAP-1 and MJAP-2 differ from each other in the number and position of a seven-amino-acid repeat and in five other amino acids. The mjap genes are expressed in the subventral pharyngeal glands of second-stage juveniles of M. javanica, and transcription analysis in different developmental stages showed expression in the juvenile stage but not in eggs or adult females. Both mjap-1 and mjap-2 were expressed in both Mi-virulent and avirulent lines of M. javanica. [ABSTRACT FROM AUTHOR]

The motility and the chemotactic response towards plant roots of Radopholus similis, after treatment with novel types of lectins, were examined in vitro by analysing movement tracks on agar plates. Six plant lectins belonging to five different lectin families and a banana thaumatin-like protein (BanTLP) were included in the experiment. A 1% concentration of Phaseolus vulgaris agglutinin (PHA) had an adverse effect on the motility of R. similis females: 63% showed no or very little movement on agar plates compared to an average of 33% for other lectins and 3% for the control treatment. A 0.05% concentration of PHA still reduced the motility of R. similis females by 75%. Concanavalin A and wheat germ agglutinin did not alter the chemotactic response towards plant roots, despite binding of both lectins to R. similis. In contrast, Galanthus nivalis agglutinin (GNA) reduced orientated movement of R. similis towards plant roots. Subsequently, secretions of R. similis were stained with Coomassie Brilliant Blue R. Nematodes treated with GNA produced secretions less abundantly compared to the control treatment and BanTLP. The other lectins in the experiment had variable effects on secretion. [ABSTRACT FROM AUTHOR]

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 L'Expressed Sequence Tag pour l'analyse de gènes s'exprimant chez les juvéniles de deuxième stade des nématodes à kyste de la pomme de terre Globodera rostochiensis et G. pallida - l'Expressed Sequence Tag (EST) ouvre une voie rapide vers la découverte de nouveaux gènes. Des gènes s'exprimant chez un large éventail de nématodes parasites d'importance médicale ou vétérinaire ont été étudiés par analyse EST alors que cette technique n'a pas encore été appliquée aux nématodes phytoparasites. Nous décrivons ici un projet EST à petite échelle utilisant les banques d'ADNc constituées à partir de deux espèces de nématodes à kyste de la pomme de terre (PCN), Globodera rostochiensis et G. pallida et nous évaluons l'utilité de cette approche pour identifier les ARNs codant les protéines abondamment sécrétées - et les autres protéines - présentes chez les nématodes lors de l'attaque parasitaire. Mille séquences environ ont été obtenues chez G. rostochiensis et 100 chez G. pallida. Des gènes variés ont été caractérisés et environ 11% des ADNc séquencés sont apparemment spécifiques des PCN. Parmi les protéines sécrétées identifiées figurent un nouvel homologue PCN de la chorismate mutase ainsi qu'un ADNc récemment cloné à partir de cellules glandulaires de Meloidogyne javanica. Les résultats ainsi obtenus justifient une utilisation à plus grande échelle de l'EST pour l'étude de ces parasites. [ABSTRACT FROM AUTHOR]

Post-fixation with osmium tetroxide (OsO[sub 4]), prior to examination by electron microscopy, enables nematodes to be located in epoxy-resin blocks, and improves contrast of ultrastructural features. For specimen visibility, alternatives to OsO[sub 4] were examined as it can inhibit antigenicity, thus preventing application of serological techniques. Basic fuchsin stain does not affect antigenicity and when applied to trichodorids enabled specimens to be readily located in the block, but had several disadvantages such as causing substantial alteration of the nematode structure. Safranin-O stain enabled Paratrichodorus anemones and P. pachydermus specimens to be located in resin blocks, different parts of the nematode body to be distinguished, and did not affect specimen ultrastructure. Also, with viruliferous specimens it allowed immunogold labelling techniques to be applied for identifying tobacco rattle virus particles at the site of retention in the nematodes. Safranin-O is fluorescent and this feature was used to examine sections from the spicula region of a male P. pachydermus specimen under a confocal laser scanning microscope. La post-fixation au tétroxyde d’osmium (OsO[sub 4]) avant l’observation en microscopie électronique empêche la localisation des nématodes dans les blocs de résine epoxy et augmente le contraste des caractéristiques ultrastructurales. Pour la localisation des spécimens, des solutions alternatives au OsO[sub 4], qui peut inhiber l’antigénicité et donc empêcher l’application de techniques sérologiques, ont été examinées. Le coloration à la fuschine basique n’affecte pas l’antigénicité et permet de situer les spécimens dans les blocs, mais elle présente de nombreux désavantages telle l’altération substantielle des structures du nématode. La safranine-O permet la localisation de Paratrichodorus anemones et de P. pachydermus, les différentes parties du corps du nématode étant alors identifiables, et n’affecte en rien l’ultrastructure du nématode. Chez les spécimens portant des virus, elle permet également l’utilisation de techniques de marquage immunologique à l’or pour identifier les particules du virus du tobacco rattle dans les sites de rétention chez le nématode. La safranine-O est fluorescente et cette particularité a été utilisée pour observer des sections de la région des spicules chez un mâle de P. pachydermus en microscopie confocale à balayage. [ABSTRACT FROM AUTHOR]