RESUMEN
Plant parasitic nematodes are microscopic pathogens that invade plant roots and cause extensive damage to crops. We have used a chemical biology approach to define mechanisms underpinning their parasitic behaviour: We discovered that reserpine, a plant alkaloid that inhibits the vesicular monoamine transporter (VMAT), potently impairs the ability of the potato cyst nematode Globodera pallida to enter the host plant root. We show this is due to an inhibition of serotonergic signalling that is essential for activation of the stylet which is used to access the host root. Prompted by this we identified core molecular components of G. pallida serotonin signalling encompassing the target of reserpine, VMAT; the synthetic enzyme for serotonin, tryptophan hydroxylase; the G protein coupled receptor SER-7 and the serotonin-gated chloride channel MOD-1. We cloned each of these molecular components and confirmed their functional identity by complementation of the corresponding C. elegans mutant thus mapping out serotonergic signalling in G. pallida. Complementary approaches testing the effect of chemical inhibitors of each of these signalling elements on discrete sub-behaviours required for parasitism and root invasion reinforce the critical role of serotonin. Thus, targeting the serotonin signalling pathway presents a promising new route to control plant parasitic nematodes.
Asunto(s)
Protección de Cultivos/métodos , Interacciones Huésped-Patógeno , Nematodos/fisiología , Enfermedades de las Plantas/parasitología , Serotonina/metabolismo , Transducción de Señal , Solanum tuberosum/metabolismo , Animales , Solanum tuberosum/parasitologíaRESUMEN
BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.
Asunto(s)
Genoma de Protozoos , Enfermedades de las Plantas/parasitología , Solanum tuberosum/parasitología , Tylenchoidea/genética , Tylenchoidea/patogenicidad , Animales , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Transferencia de Gen Horizontal , Islas Genómicas , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Estadios del Ciclo de Vida , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Sitios de Empalme de ARN , Empalme del ARN , Transcriptoma , Tylenchoidea/crecimiento & desarrollo , Virulencia/genéticaRESUMEN
Distinct populations of the potato cyst nematode (PCN) Globodera pallida exist in the UK that differ in their ability to overcome various sources of resistance. An efficient method for distinguishing between populations would enable pathogen-informed cultivar choice in the field. Science and Advice for Scottish Agriculture (SASA) annually undertake national DNA diagnostic tests to determine the presence of PCN in potato seed and ware land by extracting DNA from soil floats. These DNA samples provide a unique resource for monitoring the distribution of PCN and further interrogation of the diversity within species. We identify a region of mitochondrial DNA descriptive of three main groups of G. pallida present in the UK and adopt a metagenetic approach to the sequencing and analysis of all SASA samples simultaneously. Using this approach, we describe the distribution of G. pallida mitotypes across Scotland with field-scale resolution. Most fields contain a single mitotype, one-fifth contain a mix of mitotypes, and less than 3% contain all three mitotypes. Within mixed fields, we were able to quantify the relative abundance of each mitotype across an order of magnitude. Local areas within mixed fields are dominated by certain mitotypes and indicate towards a complex underlying 'pathoscape'. Finally, we assess mitotype distribution at the level of the individual cyst and provide evidence of 'hybrids'. This study provides a method for accurate, quantitative and high-throughput typing of up to one thousand fields simultaneously, while revealing novel insights into the national genetic variability of an economically important plant parasite.
Asunto(s)
Variación Genética , Genética de Población , Solanum tuberosum/parasitología , Tylenchoidea/genética , Animales , Código de Barras del ADN Taxonómico , ADN de Helmintos/genética , ADN Mitocondrial/genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/parasitología , Escocia , SueloRESUMEN
BACKGROUND: The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. RESULTS: The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. CONCLUSION: This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.
Asunto(s)
Genómica , Proteínas del Helminto/genética , Enfermedades de las Plantas/parasitología , Solanum tuberosum/parasitología , Tylenchoidea/genética , Tylenchoidea/fisiología , Empalme Alternativo , Animales , Femenino , Proteínas del Helminto/metabolismo , Espacio Intracelular/parasitología , Estadios del Ciclo de Vida/genética , Masculino , Solanum tuberosum/citología , Tylenchoidea/crecimiento & desarrollo , Tylenchoidea/metabolismoRESUMEN
Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.
Asunto(s)
Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/parasitología , Solanum tuberosum/genética , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Pared Celular/metabolismo , Clonación Molecular , Biología Computacional , Variaciones en el Número de Copia de ADN , ADN de Helmintos/genética , Proteínas del Helminto/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Immunoblotting , Hibridación in Situ , Estadios del Ciclo de Vida/genética , Datos de Secuencia Molecular , Familia de Multigenes , Células Vegetales/metabolismo , Enfermedades de las Plantas/genética , Raíces de Plantas/química , Raíces de Plantas/parasitología , Infecciones por Secernentea/genética , Infecciones por Secernentea/metabolismo , Infecciones por Secernentea/parasitología , Homología de Secuencia de Aminoácido , Solanum tuberosum/citología , Solanum tuberosum/parasitología , Tylenchoidea/crecimiento & desarrollo , Tylenchoidea/metabolismoRESUMEN
⢠Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. ⢠Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. ⢠Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. ⢠This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.
Asunto(s)
Arabidopsis/citología , Arabidopsis/parasitología , Pared Celular/metabolismo , Células Gigantes/parasitología , Raíces de Plantas/citología , Raíces de Plantas/parasitología , Tylenchoidea/fisiología , Animales , Epítopos/inmunología , Esterificación , Conducta Alimentaria/fisiología , Femenino , Células Gigantes/citología , Glucanos/metabolismo , Mananos/inmunología , Pectinas/metabolismo , Enfermedades de las Plantas/parasitología , Polisacáridos/metabolismo , Xilanos/metabolismo , Xilema/citología , Xilema/parasitologíaRESUMEN
Current and future global crop yields depend upon soil quality to which soil organisms make an important contribution. The European Union seeks to protect European soils and their biodiversity for instance by amending its Directive on pesticide usage. This poses a challenge for control of Globodera pallida (a potato cyst nematode) for which both natural resistance and rotational control are inadequate. One approach of high potential is transgenically based resistance. This work demonstrates the potential in the field of a new transgenic trait for control of G. pallida that suppresses root invasion. It also investigates its impact and that of a second transgenic trait on the non-target soil nematode community. We establish that a peptide that disrupts chemoreception of nematodes without a lethal effect provides resistance to G. pallida in both a containment and a field trial when precisely targeted under control of a root tip-specific promoter. In addition we combine DNA barcoding and quantitative PCR to recognise nematode genera from soil samples without microscope-based observation and use the method for nematode faunal analysis. This approach establishes that the peptide and a cysteine proteinase inhibitor that offer distinct bases for transgenic plant resistance to G. pallida do so without impact on the non-target nematode soil community.
Asunto(s)
Enfermedades de las Plantas/prevención & control , Plantas Modificadas Genéticamente , Solanum tuberosum/parasitología , Animales , Inhibidores de Cisteína Proteinasa/farmacología , Nematodos , Péptidos/farmacología , Plaguicidas , Enfermedades de las Plantas/parasitología , Suelo/normas , TylenchoideaRESUMEN
The potential of the MDK4-20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode-repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root-specific ß-glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED-E5 established that the MDK4-20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode-repellent peptide under the control of either AtMDK4-20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4-20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4-20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4-20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst-nematode defence.
Asunto(s)
Arabidopsis/genética , Nematodos/efectos de los fármacos , Péptidos/genética , Control de Plagas/métodos , Regiones Promotoras Genéticas , Solanum tuberosum/genética , Animales , Ingeniería Genética , Proteínas Fluorescentes Verdes/análisis , Péptidos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/parasitología , Plantas Modificadas Genéticamente/metabolismo , Solanum tuberosum/parasitologíaRESUMEN
In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.
Asunto(s)
Etiquetas de Secuencia Expresada , Proteínas del Helminto/fisiología , Solanum tuberosum/parasitología , Tylenchoidea/metabolismo , Tylenchoidea/patogenicidad , Animales , Biología Computacional , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Tylenchoidea/genéticaRESUMEN
Migration of plant-parasitic nematode infective larval stages through soil and invasion of roots requires perception and integration of sensory cues culminating in particular responses that lead to root penetration and parasite establishment. Components of the chemoreceptive neuronal circuitry involved in these responses are targets for control measures aimed at preventing infection. Here we report, to our knowledge, the first isolation of cyst nematode ace-2 genes encoding acetylcholinesterase (AChE). The ace-2 genes from Globodera pallida (Gp-ace-2) and Heterodera glycines (Hg-ace-2) show homology to ace-2 of Caenorhabditis elegans (Ce-ace-2). Gp-ace-2 is expressed most highly in the infective J2 stage with lowest expression in the early parasitic stages. Expression and functional analysis of the Globodera gene were carried out using the free-living nematode C. elegans in order to overcome the refractory nature of the obligate parasite G. pallida to many biological studies. Caenorhabditis elegans transformed with a GFP reporter construct under the control of the Gp-ace-2 promoter exhibited specific and restricted GFP expression in neuronal cells in the head ganglia. Gp-ACE-2 protein can functionally complement its C. elegans homologue. A chimeric construct containing the Ce-ace-2 promoter region and the Gp-ace-2 coding region and 3' untranslated region was able to restore a normal phenotype to the uncoordinated C. elegans double mutant ace-1;ace-2. This study demonstrates conservation of AChE function and expression between free-living and plant-parasitic nematode species, and highlights the utility of C. elegans as a heterologous system to study neuronal aspects of plant-parasitic nematode biology.
Asunto(s)
Acetilcolinesterasa/genética , Caenorhabditis elegans/genética , Homología de Secuencia de Ácido Nucleico , Tylenchoidea/genética , Acetilcolinesterasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia Conservada/genética , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Genoma de los Helmintos , Proteínas Fluorescentes Verdes , Locomoción/fisiología , Masculino , Datos de Secuencia Molecular , Fenotipo , Regiones Promotoras Genéticas , Alineación de Secuencia , Análisis de Secuencia de ADN , Solanum tuberosum/parasitología , Tylenchoidea/enzimología , Tylenchoidea/crecimiento & desarrolloRESUMEN
An aminopeptidase full-length cDNA (Hg-amp-1) was cloned from the adult female soybean cyst nematode Heterodera glycines by heterologous screening of a cDNA library with a Caenorhabditis elegans EST sequence. The predicted open reading frame encoded an 882-amino acid protein containing the conserved zinc-binding domain and GAMEN motif that are characteristic of M1 family aminopeptidases. The putative protein lacks any subcellular targeting signals and displays strong similarity to puromycin-sensitive aminopeptidases from C. elegans, Drosophila and mammals. Hg-amp-1 is expressed in juvenile nematodes and both male and female adults, with highest expression in gravid females. In situ mRNA hybridisation localised the Hg-amp-1 transcript to the genital primordium of pre-parasitic juvenile nematodes and the reproductive tract of adult females. Suppression of Hg-amp-1 transcript level by RNA-interference led to a 61% reduction in the number of female nematodes parasitising soybean roots 21 days post infection with infective juvenile nematodes that had been exposed to double-stranded RNA.