RESUMO
The rice root-knot nematode, Meloidogyne graminicola, is a serious pest in most rice-growing countries. Usually, nematodes employ antioxidants to counteract the harm of reactive oxygen species (ROS) and facilitate their infection. Here the gene encoding M. graminicola protein disulphide isomerase (MgPDI) was identified. The deduced protein is highly conserved in the putative active-site Cys-Gly-His-Cys. In situ hybridization showed that MgPDI was specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2s). MgPDI was significantly up-regulated in the late parasitic J2s. Characterization of the recombinant protein showed that the purified MgPDI exhibited similar activities to other oxidases/isomerases such as the refolding of the scrambled RNase and insulin disulfide reductase and the protection of plasmid DNA and living cells from ROS damage. In addition, silencing of MgPDI by RNA interference in the pre-parasitic J2s lowered their multiplication factor. MgPDI expression was up-regulated in the presence of exogenous H2O2, whereas MgPDI silencing resulted in an increase in mortality under H2O2 stress. MgPDI is localized in the apoplast when transient expression in Nicotiana benthamiana leaves. The results indicated that MgPDI plays important roles in the reproduction and pathogenicity of M. graminicola and it also contributes to protecting nematodes from exogenous H2O2 stress.
Assuntos
Proteínas de Helminto/química , Peróxido de Hidrogênio/química , Oryza/parasitologia , Isomerases de Dissulfetos de Proteínas/química , Tylenchoidea/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sequência Conservada , Expressão Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Cinética , Modelos Moleculares , Estresse Oxidativo , Doenças das Plantas/parasitologia , Raízes de Plantas/parasitologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Nicotiana/parasitologia , Tylenchoidea/enzimologia , Tylenchoidea/patogenicidadeRESUMO
Plant parasitic nematode infection of crops can be highly detrimental to agricultural production. Since the discovery that plant roots release chemicals that attract the infective stage of plant parasitic nematodes some 80 years ago, significant progress in identifying the signaling molecules has occurred only relatively recently. Here, we review the literature on chemical ecological studies of two major plant parasitic nematode groups: root knot nematodes in the genus Meloidogyne and cyst nematodes in the genus Globodera because of the negative impact their parasitism has on farming systems in Africa. We then highlight perspectives for future directions for their management.
Assuntos
Doenças das Plantas/parasitologia , Plantas/parasitologia , Tylenchoidea/química , Animais , Interações Hospedeiro-Parasita , Filogenia , Doenças das Plantas/prevenção & controle , Plantas/química , Tylenchoidea/genética , Tylenchoidea/fisiologiaRESUMO
Root-knot nematodes (RKNs) are highly specialized parasites that interact with their host plants using a range of strategies. The esophageal glands are the main places where nematodes synthesize effector proteins, which play central roles in successful invasion. The Meloidogyne incognita effector MiISE5 is exclusively expressed within the subventral esophageal cells and is upregulated during early parasitic stages. In this study, we show that MiISE5 can be secreted to barley cells through infectious hyphae of Magnaporthe oryzae. Transgenic Arabidopsis plants expressing MiISE5 became significantly more susceptible to M. incognita. Inversely, the tobacco rattle virus (TRV)-mediated silence of MiISE5 decreased nematode parasitism. Moreover, transient expression of MiISE5 suppressed cell death caused by Burkholderia glumae in Nicotiana benthamiana. Based on transcriptome analysis of MiISE5 transgenic sample and the wild-type (WT) sample, we obtained 261 DEGs, and the results of GO and KEGG enrichment analysis indicate that MiISE5 can interfere with various metabolic and signaling pathways, especially the JA signaling pathway, to facilitate nematode parasitism. Results from the present study suggest that MiISE5 plays an important role during the early stages of parasitism and provides evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by M. incognita.
Assuntos
Arabidopsis/genética , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/genética , Doenças das Plantas/genética , Sequência de Aminoácidos/genética , Animais , Apoptose/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/parasitologia , Perfilação da Expressão Gênica , Proteínas de Helminto/biossíntese , Doenças das Plantas/parasitologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/parasitologia , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/parasitologia , Tylenchoidea/química , Tylenchoidea/genéticaRESUMO
Pot and greenhouse trials were conducted for the management of root-knot nematode, Meloidogyne javanica, infestation in tomato. Growth parameters, gall index, soil, and root nematode populations were measured to assess the effect of a novel bio-pesticide (Dazitol®), made from mustard oil and oleoresin of Capsicum, on plant growth and nematode reproduction. Data generated within the pot experiment showed that the tested bio-pesticide did not improve plant growth, but it reduced significantly root-knot nematode damage resulting in a decrease in gall index and root (91%) and soil (62%) population of M. javanica compared with untreated plants. The greenhouse experiment showed that Mocap® and Dazitol® decreased nematode incidence significantly (P < 0.05) on tomato. The result of this study suggested that the best nematode control was obtained by combining soil solarization with chemical or botanical nematicides as an integrated pest management approach.
Assuntos
Antinematódeos/química , Solanum lycopersicum/crescimento & desenvolvimento , Tylenchoidea/química , Animais , Produtos Biológicos , Capsicum , Solanum lycopersicum/química , Controle de Pragas , SoloRESUMO
Plant parasitic nematodes infect a wide range of hosts representing the largest source of biotic stress experienced by plants. Meloidogyne genus comprises the most important parasitic nematodes, also known as root-knot nematodes. These parasitic organisms obtain nutrients to support their development through complex interactions with their hosts. The translationally-controlled tumor protein (TCTP) is widely expressed in eukaryotic organisms, and is related to a great diversity of biological processes such as calcium binding, cell proliferation and growth, pluripotency, regulation of apoptosis, microtubules stabilization, and histamine release. TCTP has been identified in the secretions of plant-parasitic nematodes, and may play a role in suppressing the plant immunity and programmed cell, hence promoting nematode parasitism. Our results revealed a high conservation of the evaluated protein sequences and little variation in their physico-chemical characteristics, such as isoelectric points and hidropathicity. Phylogenetic analysis also revealed the presence of three main groups of TCTPs, corresponding to plant parasitic, animal parasitic and free-living nematodes. Six plant parasitic TCTPs tertiary structure models were generated by homology modeling. The constructed models were highly similar and most of the structural variations occurred outside the characterized functional domains. To our knowledge, these are the first theoretical models of plant parasitic nematodes TCTPs and these results may provide a theoretical basis for future studies of host plant resistance to nematode infection.
Assuntos
Biomarcadores Tumorais/química , Proteínas de Helminto/química , Animais , Sequência Conservada , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Homologia de Sequência , Proteína Tumoral 1 Controlada por Tradução , Tylenchoidea/química , Tylenchoidea/genética , Tylenchoidea/metabolismoRESUMO
BACKGROUND: Macrocyclic lactones are arguably the most successful chemical class with efficacy against parasitic nematodes. Here we investigated the effect of the macrocyclic lactone ivermectin on lipid homeostasis in the plant parasitic nematode Globodera pallida and provide new insight into its mode of action. RESULTS: A non-invasive, non-destructive, label-free and chemically selective technique called Coherent anti-Stokes Raman scattering (CARS) spectroscopy was used to study lipid stores in G. pallida. We optimised the protocol using the free-living nematode Caenorhabditis elegans and then used CARS to quantify lipid stores in the pre-parasitic, non-feeding J2 stage of G. pallida. This revealed a concentration of lipid stores in the posterior region of J2 s within 24 h of hatching which decreased to undetectable levels over the course of 28 days. We tested the effect of ivermectin on J2 viability and lipid stores. Within 24 h, ivermectin paralysed J2 s. Counterintuitively, over the same time-course ivermectin increased the rate of depletion of J2 lipid, suggesting that in ivermectin-treated J2 s there is a disconnection between the energy requirements for motility and metabolic rate. This decrease in lipid stores would be predicted to negatively impact on J2 infective potential. CONCLUSION: These data suggest that the benefit of macrocyclic lactones as seed treatments may be underpinned by a multilevel effect involving both neuromuscular inhibition and acceleration of lipid metabolism. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Caenorhabditis elegans/química , Inseticidas/farmacologia , Ivermectina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Análise Espectral Raman/métodos , Tylenchoidea/efeitos dos fármacos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Lipídeos/química , Tylenchoidea/química , Tylenchoidea/metabolismoRESUMO
Owing to the current deficiencies in chemical control options and unavailability of novel management strategies, root-knot nematode (M. incognita) infections remain widespread with significant socio-economic impacts. Helminth nervous systems are peptide-rich and appear to be putative drug targets that could be exploited by antihelmintic chemotherapy. Herein, to characterize the novel peptidergic neurotransmitters, in silico mining of M. incognita genomic and transciptomic datasets revealed the presence of 16 neuropeptide-like protein (nlp) genes with structural hallmarks of neuropeptide preproproteins; among which 13 nlps were PCR-amplified and sequenced. Two key nlp genes (Mi-nlp-3 and Mi-nlp-12) were localized to the basal bulb and tail region of nematode body via in situ hybridization assay. Mi-nlp-3 and Mi-nlp-12 were greatly expressed (in qRT-PCR assay) in the pre-parasitic juveniles and adult females, suggesting the association of these genes in host recognition, development and reproduction of M. incognita. In vitro knockdown of Mi-nlp-3 and Mi-nlp-12 via RNAi demonstrated the significant reduction in attraction and penetration of M. incognita in tomato root in Pluronic gel medium. A pronounced perturbation in development and reproduction of NLP-silenced worms was also documented in adzuki beans in CYG growth pouches. The deleterious phenotypes obtained due to NLP knockdown suggests that transgenic plants engineered to express RNA constructs targeting nlp genes may emerge as an environmentally viable option to manage nematode problems in crop plants.
Assuntos
Genes de Helmintos , Neuropeptídeos/genética , Doenças das Plantas/parasitologia , Plantas/parasitologia , Interferência de RNA , Infecções por Secernentea/parasitologia , Tylenchoidea/genética , Sequência de Aminoácidos , Animais , Genômica , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Tylenchoidea/química , Tylenchoidea/fisiologia , Tylenchoidea/ultraestruturaRESUMO
A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.
Assuntos
Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Proteínas de Ligação ao Retinol/isolamento & purificação , Tylenchoidea/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/química , DNA de Helmintos/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Hibridização In Situ , Ligantes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Alinhamento de Sequência , Transcrição Gênica , Triticum/parasitologia , Tylenchoidea/genéticaRESUMO
A series of aryl hydrazones were synthesized and in vitro assayed for their activity on the root-knot nematode Meloidogyne incognita. The phenylhydrazones of thiophene-2-carboxyaldehyde 5, 3-methyl-2-thiophenecarboxyaldehyde, 6, and salicylaldehyde, 2, were the most potent with EC50/48h values of 16.6 ± 2.2, 23.2 ± 2.7, and 24.3 ± 1.4 mg/L, respectively. A GC-MS metabolomics analysis, after in vitro nematode treatment with hydrazone 6 at 100 mg/L for 12 h, revealed elevated levels of fatty acids such as lauric acid, stearic acid, 2-octenoic acid, and palmitic acid. Whereas control samples showed the highest levels of monoacylglycerols such as monostearin and 2-monostearin, surprisingly, 2 h after treatment with hydrazone 6, nematodes excreted 3 times the levels of ammonia eliminated in the same conditions by controls. Thus, phenylhydrazones may represent a good scaffold in the discovery and synthesis of new nematicidal compounds, and a metabolomics approach may be helpful in understanding their mechanisms of toxicity and mode of action.
Assuntos
Antinematódeos/farmacologia , Hidrazonas/farmacologia , Tylenchoidea/efeitos dos fármacos , Tylenchoidea/metabolismo , Animais , Antinematódeos/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hidrazonas/química , Metabolômica , Tylenchoidea/químicaRESUMO
Sedentary plant-parasitic nematodes engage in complex interactions with their host plants by secreting effector proteins. Some effectors of both root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera and Globodera spp.) mimic plant ligand proteins. Most prominently, cyst nematodes secrete effectors that mimic plant CLAVATA3/ESR-related (CLE) ligand proteins. However, only cyst nematodes have been shown to secrete such effectors and to utilize CLE ligand mimicry in their interactions with host plants. Here, we document the presence of ligand-like motifs in bona fide root-knot nematode effectors that are most similar to CLE peptides from plants and cyst nematodes. We have identified multiple tandem CLE-like motifs conserved within the previously identified Meloidogyne avirulence protein (MAP) family that are secreted from root-knot nematodes and have been shown to function in planta. By searching all 12 MAP family members from multiple Meloidogyne spp., we identified 43 repetitive CLE-like motifs composing 14 unique variants. At least one CLE-like motif was conserved in each MAP family member. Furthermore, we documented the presence of other conserved sequences that resemble the variable domains described in Heterodera and Globodera CLE effectors. These findings document that root-knot nematodes appear to use CLE ligand mimicry and point toward a common host node targeted by two evolutionarily diverse groups of nematodes. As a consequence, it is likely that CLE signaling pathways are important in other phytonematode pathosystems as well.
Assuntos
Motivos de Aminoácidos , Proteínas de Helminto/química , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Helminto/genética , Hibridização In Situ , Ligantes , Dados de Sequência Molecular , Família Multigênica , RNA Mensageiro/genética , Alinhamento de Sequência , Transdução de Sinais , Tylenchoidea/química , Tylenchoidea/genéticaRESUMO
The potato cyst nematode Globodera rostochiensis is a biotrophic pathogen that secretes effector proteins into host root cells to promote successful plant parasitism. In addition to the role in generating within root tissue the feeding cells essential for nematode development, (1) nematode secreted effectors are becoming recognized as suppressors of plant immunity. (2)(-) (4) Recently we reported that the effector ubiquitin carboxyl extension protein (GrUBCEP12) from G. rostochiensis is processed into free ubiquitin and a 12-amino acid GrCEP12 peptide in planta. Transgenic potato lines overexpressing the derived GrCEP12 peptide showed increased susceptibility to G. rostochiensis and to an unrelated bacterial pathogen Streptomyces scabies, suggesting that GrCEP12 has a role in suppressing host basal defense or possibly pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) during the parasitic interaction. (3) To determine if GrCEP12 functions as a PTI suppressor we evaluated whether GrCEP12 suppresses flg22-induced PTI responses in Nicotiana benthamiana. Interestingly, we found that transient expression of GrCEP12 in N. benthamiana leaves suppressed reactive oxygen species (ROS) production and the induction of two PTI marker genes triggered by the bacterial PAMP flg22, providing direct evidence that GrCEP12 indeed has an activity in PTI suppression.
Assuntos
Flagelina/imunologia , Nicotiana/imunologia , Nicotiana/parasitologia , Peptídeos/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Receptores de Reconhecimento de Padrão/metabolismo , Tylenchoidea/química , Animais , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Solanum tuberosum/parasitologia , Nicotiana/efeitos dos fármacos , Nicotiana/genéticaRESUMO
Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.
Assuntos
Diamida/toxicidade , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inseticidas/toxicidade , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Dados de Sequência Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Alinhamento de Sequência , Tylenchoidea/química , Tylenchoidea/efeitos dos fármacos , Tylenchoidea/genética , Tylenchoidea/metabolismoRESUMO
For the first time, we demonstrate the feasibility for the use of MALDI-TOF MS for rapid, direct and sensitive detection of single adult root-knot nematode, Meloidogyne incognita and their second stage juvenile (J(2)). We have proposed simple pretreatment protocols and have demonstrated that the crushed and washed nematodes yielded better spectra. We also report the differentiation between the harmless and harmful stages of the nematode based on mass spectrometric profiling. Peaks at m/z 4350, 4692, 4933, 8725 were only present in the adult stages, while m/z 3220, 3433, 3485, 3830, 6540, 7444, 7770 were unique to the J(2) infective stage. The only common peak to both the phases was at m/z 3277. Thus, we show that MALDI-TOF MS can be used to differentiate between the infective and non-infective stage of the nematode and the detection sensitivity of MALDI-MS could be applied to a single nematode analysis.
Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tylenchoidea/isolamento & purificação , Animais , Feminino , Estágios do Ciclo de Vida , Fatores de Tempo , Tylenchoidea/química , Tylenchoidea/crescimento & desenvolvimentoRESUMO
Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.
Assuntos
FMRFamida/química , Proteínas de Helminto/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/parasitologia , Rabditídios/enzimologia , Tylenchida/enzimologia , Tylenchoidea/enzimologia , Animais , Biocatálise , Capsicum/parasitologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/química , Cinética , Peptídeo Hidrolases/química , Rabditídios/química , Glycine max/parasitologia , Tylenchida/química , Tylenchoidea/químicaRESUMO
MOTIVATION: Identification of conserved motifs in biological sequences is crucial to unveil common shared functions. Many tools exist for motif identification, including some that allow degenerate positions with multiple possible nucleotides or amino acids. Most efficient methods available today search conserved motifs in a set of sequences, but do not check for their specificity regarding to a set of negative sequences. RESULTS: We present a tool to identify degenerate motifs, based on a given classification of amino acids according to their physico-chemical properties. It returns the top K motifs that are most frequent in a positive set of sequences involved in a biological process of interest, and absent from a negative set. Thus, our method discovers discriminative motifs in biological sequences that may be used to identify new sequences involved in the same process. We used this tool to identify candidate effector proteins secreted into plant tissues by the root knot nematode Meloidogyne incognita. Our tool identified a series of motifs specifically present in a positive set of known effectors while totally absent from a negative set of evolutionarily conserved housekeeping proteins. Scanning the proteome of M. incognita, we detected 2579 proteins that contain these specific motifs and can be considered as new putative effectors. AVAILABILITY AND IMPLEMENTATION: The motif discovery tool and the proteins used in the experiments are available at http://dtai.cs.kuleuven.be/ml/systems/merci.
Assuntos
Algoritmos , Motivos de Aminoácidos , Biologia Computacional/métodos , Sequência Conservada , Proteínas de Helminto/química , Sequência de Aminoácidos , Animais , Análise Discriminante , Proteínas de Helminto/classificação , Dados de Sequência Molecular , Proteoma/análise , Alinhamento de Sequência , Tylenchoidea/químicaRESUMO
FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.
Assuntos
FMRFamida/metabolismo , Proteínas de Helminto/metabolismo , Doenças das Plantas/parasitologia , Tylenchoidea/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , FMRFamida/química , FMRFamida/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Solanum lycopersicum/parasitologia , Dados de Sequência Molecular , Raízes de Plantas/parasitologia , Alinhamento de Sequência , Tylenchoidea/química , Tylenchoidea/genéticaRESUMO
The method introduced in this article makes use of the glutaraldehyde-induced auto-fluorescence of proteins after cross-linking with glutaraldehyde for the analysis of cellular and sub-cellular structures. Because the interface of biotrophic interactions is rich in proteins, the method presented is particularly suitable for the analysis of such interactions; we have exemplified its usefulness by analyzing (1) the root feeding sites induced in roots from Arabidopsis thaliana by the root-knot nematode Meloidogyne incognita; (2) leaves from Cucurbita pepo infected by powdery mildew and (3) roots from Nicotiana tabacum colonized by the arbuscular mycorrhizal fungus Glomus intraradices. The use of confocal and multi-photon laser scanning microscopy allows three-dimensional reconstructions from optical sections of complex biotrophic interactions. In the case of root-knot nematode feeding sites, our method enabled us to simultaneously study the development of the plant xylem elements (using lignin auto-fluorescence), the nematode feeding site and the nematode itself.
Assuntos
Arabidopsis/parasitologia , Cucurbita/microbiologia , Fluorescência , Fungos/química , Glutaral/metabolismo , Microscopia de Fluorescência/métodos , Nicotiana/microbiologia , Tylenchoidea/química , Animais , Proteínas Fúngicas/análise , Fungos/crescimento & desenvolvimento , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Proteínas/análise , Tylenchoidea/crescimento & desenvolvimentoRESUMO
A new venom allergen-like protein gene isolated from Meloidogyne incognita (designated Mi-vap-2) was cloned and analysed. The genomic clone of Mi-vap-2 is 1917-bp long, contains three introns, which range in size from 39 to 797 bp, and four exons ranging in size from 37 to 361 bp. The cDNA of Mi-vap-2 contains an open reading frame encoding 294 amino acids, being the first 16 residues a putative secretion signal. Southern blot analysis suggested that Mi-vp-2 is probably a member of a small multigene family. In situ hybridization analysis showed that the transcripts of Mi-vap-2 accumulated exclusively within the subventral oesophageal gland cells of M. incognita. RT-PCR analyses confirmed that Mi-vap-2 was transcribed mainly in the pre-parasitic second-stage and early post-inoculated juveniles. Results indicated that this venom allergen-like protein gene may play an important role in establishment of the parasitic relationship between plants and nematodes.
Assuntos
Antígenos de Helmintos/genética , Proteínas de Helminto/genética , Tylenchoidea/genética , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA de Helmintos/química , DNA de Helmintos/genética , Feminino , Proteínas de Helminto/química , Hibridização In Situ , Solanum lycopersicum/parasitologia , Dados de Sequência Molecular , Raízes de Plantas/parasitologia , Reação em Cadeia da Polimerase , RNA de Helmintos/genética , RNA Mensageiro/genética , Alinhamento de Sequência , Tylenchoidea/química , Tylenchoidea/imunologiaRESUMO
Sequence variability and distribution of a newly characterized MPA2 satellite DNA family are described in five root-knot nematode species of the genus Meloidogyne, the mitotic parthenogens M. paranaensis, M. incognita, M. arenaria and M. javanica, and the meiotic/mitotic M. hapla (isolates A and B, respectively). The lack of distinctive mutations and the considerable contribution (40.8%) of ancestral changes disclose an ancient satellite DNA which existed in the common ancestor of extant parthenogenetic species in the same or similar form and remained preserved for a period of at least 43 My. Nonuniformly distributed polymorphic sites along the satellite monomer suggest differences in constraints acting on particular sequence segments. Sequence diversity is clearly unaffected by significant differences in genomic abundance of the MPA2 satellite DNA in the examined species. Observed results suggest that the dynamics of this satellite DNA family might be in the first instance a consequence of characteristics of its nucleotide sequence and possible constraints imposed on it. Under conditions of mitotic and meiotic parthenogenesis, slow accumulation of mutations and slow replacement of old MPA2 sequence variants with new ones may be equivalent to the dynamics of some satellite DNA sequences conserved for extremely long evolutionary periods in sexual species.
Assuntos
DNA de Helmintos/genética , DNA Satélite/química , Amplificação de Genes , Partenogênese , Tylenchoidea/genética , Animais , Sequência de Bases , DNA de Helmintos/química , DNA Satélite/genética , Evolução Molecular , Variação Genética , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Tylenchoidea/químicaRESUMO
Two satellite DNAs have been characterized in the mitotic parthenogenetic root-knot nematodes Meloidogyne javanica and M. paranaensis, agriculturally important phytoparasitic species. The satellite repeat variants cloned from M. javanica could not be resolved from those described earlier in M. arenaria [Castagnone-Sereno, P., Leroy, F., Abad, P., 2000. Cloning and characterization of an extremely conserved satellite DNA family from the root-knot nematode Meloidogyne arenaria. Genome 43, 346-353] and are therefore classified as a single satellite named MARJA. However, this satellite shows 34.3% sequence divergence in comparison with the MPA1 satellite characterized in M. paranaensis, and monomer variants of both satellites are clearly distinguished by homogenized nucleotide substitutions. Nucleotide variability analysis revealed in one segment of the satellite monomer domains of high and low variability, conserved both within and between monomer variants of the two satellites. Intersatellite conservation of these domains indicates evolution of satellite sequence under different constraints, probably due to some functional interactions. In addition, high intrasatellite homogeneity, presence of ancestral mutations in groups of MARJA monomers in both M. javanica and M. arenaria and highly homogenized divergent positions in comparison with the MPA1 indicate similar sequence dynamics in mitotic parthenogenetic taxa to that observed in amphimictic species.