RESUMO
Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.
Assuntos
Proteínas de Plantas/genética , Solanum tuberosum/genética , Sequência de Bases , Southern Blotting , Primers do DNA , Família Multigênica , Solanum tuberosum/parasitologia , Solanum tuberosum/virologiaAssuntos
Polissacarídeo-Liases/metabolismo , Tylenchida/fisiologia , Sequência de Aminoácidos , Animais , Parede Celular/parasitologia , Clonagem Molecular , Dados de Sequência Molecular , Pectinas/metabolismo , Poligalacturonase/genética , Poligalacturonase/metabolismo , Polissacarídeo-Liases/classificação , Polissacarídeo-Liases/genética , Tylenchida/enzimologia , Tylenchida/genéticaRESUMO
Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.
Assuntos
Adenina/análogos & derivados , Leucócitos Mononucleares/citologia , Ácidos Naftalenoacéticos/farmacologia , Nematoides/fisiologia , Nicotiana/citologia , Reguladores de Crescimento de Plantas/farmacologia , Plantas Tóxicas , Solanum tuberosum/parasitologia , Adenina/farmacologia , Animais , Compostos de Benzil , Divisão Celular , Humanos , Cinetina , Leucócitos Mononucleares/efeitos dos fármacos , Folhas de Planta , Protoplastos/efeitos dos fármacos , Protoplastos/fisiologia , Purinas , Nicotiana/efeitos dos fármacos , Nicotiana/fisiologiaRESUMO
Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Grânulos Citoplasmáticos/química , Sistema Digestório/química , Proteínas de Helminto/isolamento & purificação , Nematoides/química , Animais , Anticorpos Monoclonais , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Reações Cruzadas , Grânulos Citoplasmáticos/ultraestrutura , Sistema Digestório/ultraestrutura , Imunofluorescência , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Microscopia Imunoeletrônica , Nematoides/crescimento & desenvolvimento , Nematoides/patogenicidade , Nematoides/ultraestrutura , Solanum tuberosum/parasitologia , Especificidade da Espécie , VirulênciaRESUMO
Random amplified polymorphic DNA (RAPD) offers a potential basis for the development of a diagnostic assay to differentiate the potato cyst nematode species Globodera rostochiensis and G. pallida. Nine decamer primers have been tested for their ability to amplify species-specific DNA sequences. Primer OPG-05 produced 2 discrete DNA fragments, which were consistently present in 5 G. rostochiensis populations and absent in 5 G. pallida populations. These fragments were detectable in single females as well as in single 2nd-stage juveniles. Their amplification is extremely efficient, and reproducible over a wide range of template concentrations. One-fifth of a single juvenile is sufficient to generate reproducible RAPD markers. The amplification from single juveniles requires no DNA isolation. The use of a crude homogenate does not impair the polymerase chain reaction.