RESUMEN
KEY MESSAGE: Functional characterization of the Columbia root-knot nematode resistance gene R Mc1 ( blb ) in potato revealed the R gene-mediated resistance is dependent on a hypersensitive response and involves calcium. The resistance (R) gene R Mc1(blb) confers resistance against the plant-parasitic nematode, Meloidogyne chitwoodi. Avirulent and virulent nematodes were used to functionally characterize the R Mc1(blb)-mediated resistance mechanism in potato (Solanum tuberosum). Histological observations indicated a hypersensitive response (HR) occurred during avirulent nematode infection. This was confirmed by quantifying reactive oxygen species activity in response to avirulent and virulent M. chitwoodi. To gain an insight into the signal transduction pathways mediating the R Mc1(blb)-induced HR, chemical inhibitors were utilized. Inhibiting Ca(2+) channels caused a significant reduction in electrolyte leakage, an indicator of cell death. Labeling with a Ca(2+)-sensitive dye revealed high Ca(2+) levels in the root cells surrounding avirulent nematodes. Furthermore, the calcium-dependent protein kinase (CDPK), StCDPK4 had a higher transcript level in R Mc1(blb) potato roots infected with avirulent nematodes in comparison to roots infected with virulent M. chitwoodi. The results of this study indicate Ca(2+) plays a role in the R Mc1(blb)-mediated resistance against M. chitwoodi in potato.
Asunto(s)
Calcio/inmunología , Resistencia a la Enfermedad/inmunología , Genes de Plantas/inmunología , Enfermedades de las Plantas/inmunología , Solanum tuberosum/inmunología , Tylenchoidea/inmunología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Resistencia a la Enfermedad/genética , Electrólitos/metabolismo , Regulación de la Expresión Génica de las Plantas/inmunología , Genes de Plantas/genética , Interacciones Huésped-Parásitos/inmunología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/inmunología , Raíces de Plantas/parasitología , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Solanum tuberosum/genética , Solanum tuberosum/parasitología , Tylenchoidea/patogenicidad , Tylenchoidea/fisiología , Virulencia/inmunologíaRESUMEN
Meloidogyne chitwoodi, a quarantine pathogen, is a significant problem in potato-producing areas worldwide. In spite of considerable genetic diversity in wild potato species, no commercial potato cultivars with resistance to M. chitwoodi are available. Nematode effector genes are essential for the molecular interactions between root-knot nematodes and their hosts. Stable transgenic lines of Arabidopsis and potato (Solanum tuberosum) with resistance against M. chitwoodi were developed. RNA interference (RNAi) construct pART27(16D10i-2) was introduced into Arabidopsis thaliana and potato to express double-stranded RNA complementary to the putative M. chitwoodi effector gene Mc16D10L. Plant-mediated RNAi led to a significant level of resistance against M. chitwoodi in Arabidopsis and potato. In transgenic Arabidopsis lines, the number of M. chitwoodi egg masses and eggs was reduced by up to 57 and 67% compared with empty vector controls, respectively. Similarly, in stable transgenic lines of potato, the number of M. chitwoodi egg masses and eggs was reduced by up to 71 and 63% compared with empty vector controls, respectively. The relative transcript level of Mc16D10L was reduced by up to 76% in M. chitwoodi eggs and infective second-stage juveniles that developed on transgenic pART27(16D10i-2) potato, suggesting that the RNAi effect is systemic and heritable in M. chitwoodi.
Asunto(s)
Arabidopsis/genética , Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/inmunología , Solanum tuberosum/genética , Tylenchoidea/fisiología , Animales , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas del Helminto/metabolismo , Enfermedades de las Plantas/microbiología , Raíces de Plantas/genética , Raíces de Plantas/inmunología , Raíces de Plantas/microbiología , Interferencia de ARN , ARN Bicatenario/genética , ARN de Planta/genética , Alineación de Secuencia , Solanum tuberosum/inmunología , Solanum tuberosum/microbiologíaRESUMEN
Cyst nematodes produce parasitism proteins that contain putative nuclear localisation signals (NLSs) and, therefore, are predicted to be imported into the nucleus of the host plant cell. The in planta localisation patterns of eight soybean cyst nematode (Heterodera glycines) parasitism proteins with putative NLSs were determined by producing these proteins as translational fusions with the GFP and GUS reporter proteins. Two parasitism proteins were found to be imported into the nuclei of onion epidermal cells as well as Arabidopsis protoplasts. One of these two parasitism proteins was further transported into the nucleoli. Mutations introduced into the NLS domains of these two proteins abolished nuclear import and caused a cytoplasmic accumulation. Furthermore, we observed active nuclear uptake for three additional parasitism proteins, however, only when these proteins were synthesised as truncated forms. Two of these proteins were further transported into nucleoli. We hypothesise that nuclear uptake and nucleolar localisation are important mechanisms for H. glycines to modulate the nuclear biology of parasitised cells of its host plant.