RESUMO
Herein we describe the cloning and characterization of a cDNA encoding an aspartic proteinase from the root-knot nematode Meloidogyne incognita. Using PCR techniques, a 1471-bp cDNA fragment encoding a cathepsin D-like (Mi-asp1) transcript was isolated from second-stage larvae mRNA. Its predicted amino acid sequence comprises a pro-region of 71 amino acid residues and a mature protease of 378 amino acid residues with a predicted molecular mass of 41.502kDa. Protein sequence comparisons of Mi-asp1 with GenBank (DQ360827) sequences showed 59-71% identity with nematode-specific cathepsin D-like aspartic proteinases. Southern blot analysis, RT-PCR amplification and EST mining suggest the existence of a developmentally expressed gene family encoding aspartic proteinases in M. incognita. Mi-asp1 may represent a potential target for molecular intervention for the purposes of plant-parasitic nematode control.
Assuntos
Ácido Aspártico Endopeptidases/genética , DNA Complementar/química , DNA de Helmintos/química , Tylenchoidea/enzimologia , Tylenchoidea/genética , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Southern Blotting , Clonagem Molecular , Análise por Conglomerados , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Larva/enzimologia , Larva/genética , Solanum lycopersicum/parasitologia , Dados de Sequência Molecular , Óvulo/enzimologia , Raízes de Plantas/parasitologia , RNA de Helmintos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.
Assuntos
Proteínas de Helminto/genética , Nicotiana/genética , Nicotiana/parasitologia , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , DNA Complementar/genética , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Contagem de Ovos de Parasitas , Fenótipo , Doenças das Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas/parasitologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Nicotiana/ultraestrutura , Tylenchoidea/genética , Tylenchoidea/crescimento & desenvolvimento , Tylenchoidea/patogenicidadeRESUMO
This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.