RESUMEN
Pectin in the primary plant cell wall is thought to be responsible for its porosity, charge density, and microfibril spacing and is the main component of the middle lamella. Plant-parasitic nematodes secrete cell wall-degrading enzymes that macerate the plant tissue, facilitating the penetration and migration within the roots. In sedentary endoparasitic nematodes, these enzymes are released only during the migration of infective juveniles through the root. Later, nematodes manipulate the expression of host plant genes, including various cell wall enzymes, in order to induce specific feeding sites. In this study, we investigated expression of two Arabidopsis pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780), together with pectic epitopes with different degrees of methylesterification in both syncytia induced by the cyst nematode Heterodera schachtii and giant cells induced by the root-knot nematode Meloidogyne incognita. We confirmed upregulation of PLL18 and PLL19 in both types of feeding sites with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Furthermore, the functional analysis of mutants demonstrated the important role of both PLL genes in the development and maintenance of syncytia but not giant cells. Our results show that both enzymes play distinct roles in different infected root tissues as well as during parasitism of different nematodes.
Asunto(s)
Arabidopsis/enzimología , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/parasitología , Polisacárido Liasas/metabolismo , Tylenchida/fisiología , Tylenchoidea/fisiología , Animales , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Femenino , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Células Gigantes/citología , Masculino , Mutagénesis Insercional , Fenotipo , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/parasitología , Brotes de la Planta/citología , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/parasitología , Polisacárido Liasas/genética , Plantones/citología , Plantones/enzimología , Plantones/genética , Plantones/parasitología , Especificidad de la Especie , Tylenchida/citología , Tylenchoidea/citología , Regulación hacia ArribaRESUMEN
Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode genes.
Asunto(s)
Marcación de Gen/métodos , Nematodos/genética , Nicotiana/parasitología , Enfermedades de las Plantas/parasitología , Virus de Plantas/genética , Interferencia de ARN , Animales , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Nematodos/virología , Raíces de Plantas/parasitología , Virus de Plantas/metabolismoRESUMEN
The cell wall is an important determinant of plant cell form. Here we define a class of Arabidopsis root hair mutants with defective cell walls. Plants homozygous for kojak (kjk) mutations initiate root hairs that rupture at their tip soon after initiation. The KJK gene was isolated by positional cloning, and its identity was confirmed by the molecular complementation of the Kjk(-) phenotype and the sequence of three kjk mutant alleles. KOJAK encodes a cellulose synthase-like protein, AtCSLD3. KOJAK/AtCSLD3 is the first member of this subfamily of proteins to be shown to have a function in cell growth. Subcellular localization of the KOJAK/AtCSLD3 protein using a GFP fusion shows that KOJAK/AtCSLD3 is located on the endoplasmic reticulum, indicating that KOJAK/AtCSLD3 is required for the synthesis of a noncellulosic wall polysaccharide. Consistent with the cell specific defect in the roots of kjk mutants, KOJAK/AtCSDL3 is preferentially expressed in hair cells of the epidermis. The Kjk(-) phenotype and the pattern of KOJAK/AtCSLD3 expression suggest that this gene acts early in the process of root hair outgrowth. These results suggest that KOJAK/AtCSLD3 is involved in the biosynthesis of beta-glucan-containing polysaccharides that are required during root hair elongation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/fisiología , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/fisiología , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , División Celular , Clonación Molecular , Secuencia Conservada , Genes Reporteros , Prueba de Complementación Genética , Homocigoto , Datos de Secuencia Molecular , Morfogénesis , Raíces de Plantas/citología , Plantas Modificadas GenéticamenteRESUMEN
A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.
Asunto(s)
Celulasa/genética , ADN Complementario/genética , ADN de Helmintos/genética , Tylenchoidea/enzimología , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/aislamiento & purificación , ADN de Helmintos/aislamiento & purificación , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes de Helminto , Masculino , Datos de Secuencia Molecular , Plantas/parasitología , Tylenchoidea/patogenicidadRESUMEN
Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs.