RESUMEN
Root-knot nematodes (RKN) (Meloidogyne spp.) represent one of the most damaging groups of plant-parasitic nematodes. They secrete effector proteins through a protrusible stylet to manipulate host cells for their benefit. Stylet-secreted effector proteins are produced within specialized secretory esophageal gland cells, one dorsal gland (DG) and two subventral glands (SvG), whose activity differ throughout the nematode life cycle. Previous gland transcriptomic profiling studies identified dozens of candidate RKN effectors but were focused on the juvenile stages of the nematode, when the SvGs are most active. We developed a new approach to enrich for the active DGs of M. incognita adult female RKN for RNA and protein extraction. Female heads were manually cut from the body, and a combination of sonication and vortexing was used to dislodge contents inside the heads. DG-enriched fractions were collected by filtering, using cell strainers. Comparative transcriptome profiling of pre-parasitic second-stage juveniles, female heads, and DG-enriched samples was conducted using RNA sequencing. Application of an established effector mining pipeline led to the identification of 83 candidate effector genes upregulated in DG-enriched samples of adult females that code for proteins with a predicted signal peptide but lack transmembrane domains or homology to proteins in the free-living nematode Caenorhabditis elegans. In situ hybridization resulted in the identification of 14 new DG-specific candidate effectors expressed in adult females. Taken together, we have identified novel candidate Meloidogyne effector genes that may have essential roles during later stages of parasitism. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Nematodos , Parásitos , Tylenchoidea , Animales , Femenino , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Plantas/genética , Perfilación de la Expresión Génica , Parásitos/genética , Caenorhabditis elegans/genética , Tylenchoidea/genética , Enfermedades de las Plantas/parasitologíaRESUMEN
Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Beta vulgaris , Quistes , Tylenchoidea , Animales , Arabidopsis/metabolismo , Beta vulgaris/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tylenchoidea/genética , Tylenchoidea/metabolismo , Azúcares/metabolismo , Raíces de Plantas/parasitología , Enfermedades de las Plantas/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Serina-Treonina QuinasasRESUMEN
Cyst nematodes induce a multicellular feeding site within roots called a syncytium. It remains unknown how root cells are primed for incorporation into the developing syncytium. Furthermore, it is unclear how CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide effectors secreted into the cytoplasm of the initial feeding cell could have an effect on plant cells so distant from where the nematode is feeding as the syncytium expands. Here we describe a novel translocation signal within nematode CLE effectors that is recognized by plant cell secretory machinery to redirect these peptides from the cytoplasm to the apoplast of plant cells. We show that the translocation signal is functionally conserved across CLE effectors identified in nematode species spanning three genera and multiple plant species, operative across plant cell types, and can traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells via a previously unknown post-translational mechanism of endoplasmic reticulum (ER) translocation. Our results uncover a mechanism of effector trafficking that is unprecedented in any plant pathogen to date, andthey illustrate how phytonematodes can deliver effector proteins into host cells and then hijack plant cellular processes for their export back out of the cell to function as external signaling molecules to distant cells.
Asunto(s)
Nematodos , Tylenchoidea , Animales , Retículo Endoplásmico , Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Péptidos , Enfermedades de las Plantas , Raíces de PlantasRESUMEN
While numerous effectors that suppress plant immunity have been identified from bacteria, fungi, and oomycete pathogens, relatively little is known for nematode effectors. Several dozen effectors have been reported from the soybean cyst nematode (SCN). Previous studies suggest that a hypersensitive response-like programmed cell death is triggered at nematode feeding sites in soybean during an incompatible interaction. However, virulent SCN populations overcome this incompatibility using unknown mechanisms. A soybean BAG6 (Bcl-2 associated anthanogene 6) gene previously reported by us to be highly up-regulated in degenerating feeding sites induced by SCN in a resistant soybean line was attenuated in response to a virulent SCN population. We show that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean defence responses during infection. Thus, we used a yeast viability assay to screen SCN effector candidates for their ability to specifically suppress GmBAG6-1-induced cell death. We identified several effectors that strongly suppressed cell death mediated by GmBAG6-1. Two effectors identified as suppressors showed direct interaction with GmBAG6-1 in yeast, suggesting that one mechanism of cell death suppression may occur through an interaction with this host protein.
Asunto(s)
Arabidopsis/inmunología , Regulación de la Expresión Génica de las Plantas , Glycine max/genética , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Tylenchoidea/fisiología , Animales , Arabidopsis/genética , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Muerte Celular , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Glycine max/parasitologíaRESUMEN
Dystonia 24 was first reported in 2000 as an autosomal dominant cause of dystonia caused by variants in the ANO3 gene. Although many adults have been described with dystonia 24, since 2014, an increasing number of children have also been reported. Dystonia 24 should also be considered in the differential of a child with unexplained dystonia.
RESUMEN
Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.
Asunto(s)
Arabidopsis/genética , Arabidopsis/parasitología , Núcleo Celular/metabolismo , Conducta Alimentaria , Regulación de la Expresión Génica de las Plantas , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos/genética , Tylenchoidea/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Genes de Plantas , Proteínas del Helminto/química , Estadios del Ciclo de Vida , Señales de Localización Nuclear , Parásitos/metabolismo , Células Vegetales/metabolismo , Hojas de la Planta/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/parasitología , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Plantones/metabolismo , Tylenchoidea/crecimiento & desarrollo , Regulación hacia ArribaRESUMEN
Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 ß subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii.
Asunto(s)
Proteínas del Helminto/metabolismo , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/metabolismo , Raíces de Plantas/parasitología , Tylenchoidea/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/parasitología , Beta vulgaris , Citoplasma/metabolismo , ADN Bacteriano/genética , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas del Helminto/química , Hibridación in Situ , Mutagénesis Insercional/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Plantas Modificadas Genéticamente , Unión Proteica , Reproducibilidad de los Resultados , Alineación de SecuenciaRESUMEN
Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than 60 H. glycines candidate effectors were identified in previous gland-cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 expressed sequence tags. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole-mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.
Asunto(s)
Glycine max/parasitología , Enfermedades de las Plantas/parasitología , Tylenchoidea/fisiología , Animales , Secuencia de Bases , Biblioteca de Genes , Células Gigantes , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Raíces de Plantas/parasitología , Análisis de Secuencia de ADNRESUMEN
Plant-parasitic cyst nematodes synthesize and secrete effector proteins that are essential for parasitism. One such protein is the 10A07 effector from the sugar beet cyst nematode, Heterodera schachtii, which is exclusively expressed in the nematode dorsal gland cell during all nematode parasitic stages. Overexpression of H. schachtii 10A07 in Arabidopsis thaliana produced a hypersusceptible phenotype in response to H. schachtii infection along with developmental changes reminiscent of auxin effects. The 10A07 protein physically associates with a plant kinase and the IAA16 transcription factor in the cytoplasm and nucleus, respectively. The interacting plant kinase (IPK) phosphorylates 10A07 at Ser-144 and Ser-231 and mediates its trafficking from the cytoplasm to the nucleus. Translocation to the nucleus is phosphorylation dependent since substitution of Ser-144 and Ser-231 by alanine resulted in exclusive cytoplasmic accumulation of 10A07. IPK and IAA16 are highly upregulated in the nematode-induced syncytium (feeding cells), and deliberate manipulations of their expression significantly alter plant susceptibility to H. schachtii in an additive fashion. An inactive variant of IPK functioned antagonistically to the wild-type IPK and caused a dominant-negative phenotype of reduced plant susceptibility. Thus, exploitation of host processes to the advantage of the parasites is one mechanism by which cyst nematodes promote parasitism of host plants.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/parasitología , Núcleo Celular/metabolismo , Interacciones Huésped-Parásitos , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Tylenchoidea/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/metabolismo , Beta vulgaris/parasitología , Ácidos Indolacéticos/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Señales de Localización Nuclear , Fosforilación , Fosfoserina/metabolismo , Enfermedades de las Plantas/parasitología , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Regulación hacia ArribaRESUMEN
Vagal nerve stimulators (VNS) are surgically implantable medical devices which are approved by the food and drug administration (FDA) for treatment of medically refractory epilepsy in children. Two children with seizures disorders presented to the pediatric otolaryngology clinic with complaints of stridor and sleep apnea following implantation of VNS devices. Both children were evaluated with flexible laryngoscopy, direct laryngoscopy and bronchoscopy. The children were noted to have contraction of their vocal folds and supraglottis and the settings of their VNS were adjusted until no further contractions were noted. Each child had resolution of their symptoms following adjustment.
Asunto(s)
Prótesis e Implantes/efectos adversos , Ruidos Respiratorios/etiología , Estimulación del Nervio Vago/instrumentación , Niño , Preescolar , Humanos , Masculino , Síndromes de la Apnea del Sueño/etiologíaRESUMEN
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.
Asunto(s)
Secuencias de Aminoácidos , Proteínas del Helminto/química , Enfermedades de las Plantas/parasitología , Tylenchoidea/fisiología , Secuencia de Aminoácidos , Animales , Proteínas del Helminto/genética , Hibridación in Situ , Ligandos , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/genética , Alineación de Secuencia , Transducción de Señal , Tylenchoidea/química , Tylenchoidea/genéticaRESUMEN
Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.
Asunto(s)
Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/parasitología , Tylenchoidea/genética , Animales , Citoplasma/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN de Helmintos/química , ADN de Helmintos/genética , Regulación de la Expresión Génica , Genes Reporteros , Proteínas del Helminto/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Solanum lycopersicum/citología , Solanum lycopersicum/parasitología , Cebollas/citología , Cebollas/parasitología , Epidermis de la Planta/citología , Epidermis de la Planta/parasitología , Raíces de Plantas/parasitología , ARN de Helminto/genética , Análisis de Secuencia de ADN , Tylenchoidea/citología , Tylenchoidea/fisiologíaRESUMEN
Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.
Asunto(s)
Proteínas del Helminto/metabolismo , Nematodos/fisiología , Parásitos/metabolismo , Animales , Interacciones Huésped-Parásitos , Células Vegetales/metabolismo , Células Vegetales/parasitologíaRESUMEN
Parasitism genes encode effector proteins that are secreted through the stylet of root-knot nematodes to dramatically modify selected plant cells into giant-cells for feeding. The Mi8D05 parasitism gene previously identified was confirmed to encode a novel protein of 382 amino acids that had only one database homolog identified on contig 2374 within the Meloidogyne hapla genome. Mi8D05 expression peaked in M. incognita parasitic second-stage juveniles within host roots and its encoded protein was limited to the subventral esophageal gland cells that produce proteins secreted from the stylet. Constitutive expression of Mi8D05 in transformed Arabidopsis thaliana plants induced accelerated shoot growth and early flowering but had no visible effects on root growth. Independent lines of transgenic Arabidopsis that expressed a double-stranded RNA complementary to Mi8D05 in host-derived RNA interference (RNAi) tests had up to 90% reduction in infection by M. incognita compared with wild-type control plants, suggesting that Mi8D05 plays a critical role in parasitism by the root-knot nematode. Yeast two-hybrid experiments confirmed the specific interaction of the Mi8D05 protein with plant aquaporin tonoplast intrinsic protein 2 (TIP2) and provided evidence that the Mi8D05 effector may help regulate solute and water transport within giant-cells to promote the parasitic interaction.
Asunto(s)
Arabidopsis/parasitología , Proteínas del Helminto/genética , Proteínas de la Membrana/metabolismo , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/metabolismo , Solanum lycopersicum/parasitología , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/fisiología , Transporte Biológico , Susceptibilidad a Enfermedades , Femenino , Flores/genética , Flores/parasitología , Flores/fisiología , Expresión Génica , Células Gigantes , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Estadios del Ciclo de Vida , Solanum lycopersicum/genética , Solanum lycopersicum/fisiología , Raíces de Plantas/genética , Raíces de Plantas/parasitología , Raíces de Plantas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/parasitología , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Mapeo de Interacción de Proteínas , Interferencia de ARN , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Tylenchoidea/crecimiento & desarrollo , Tylenchoidea/fisiología , Agua/metabolismoRESUMEN
Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant ß-1,3-endoglucanase in both yeast and plant cells, possibly to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3-5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host ß-1,3-endoglucanase in the defence response against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/parasitología , Celulasa/metabolismo , Proteínas del Helminto/metabolismo , Enfermedades de las Plantas/parasitología , Tylenchoidea/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Celulasa/genética , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Unión Proteica , Tylenchoidea/genética , Tylenchoidea/crecimiento & desarrolloRESUMEN
Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.
Asunto(s)
Biblioteca de Genes , Interacciones Huésped-Parásitos/genética , Nematodos/citología , Nematodos/genética , Nematodos/patogenicidad , Raíces de Plantas/parasitología , Animales , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/aislamiento & purificaciónRESUMEN
In this article, we present the cloning of two CLAVATA3/ESR (CLE)-like genes, HsCLE1 and HsCLE2, from the beet cyst nematode Heterodera schachtii, a plant-parasitic cyst nematode with a relatively broad host range that includes the model plant Arabidopsis. CLEs are small secreted peptide ligands that play important roles in plant growth and development. By secreting peptide mimics of plant CLEs, the nematode can developmentally reprogramme root cells for the formation of unique feeding sites within host roots for its own benefit. Both HsCLE1 and HsCLE2 encode small secreted polypeptides with a conserved C-terminal CLE domain sharing highest similarity to Arabidopsis CLEs 1-7. Moreover, HsCLE2 contains a 12-amino-acid CLE motif that is identical to AtCLE5 and AtCLE6. Like all other plant and nematode CLEs identified to date, HsCLEs caused wuschel-like phenotypes when overexpressed in Arabidopsis, and this activity was abolished when the proteins were expressed without the CLE motif. HsCLEs could also function in planta without a signal peptide, highlighting the unique, yet conserved function of nematode CLE variable domains in trafficking CLE peptides for secretion. In a direct comparison of HsCLE2 overexpression phenotypes with those of AtCLE5 and AtCLE6, similar shoot and root phenotypes were observed. Exogenous application of 12-amino-acid synthetic peptides corresponding to the CLE motifs of HsCLEs and AtCLE5/6 suggests that the function of this class of CLEs may be subject to complex endogenous regulation. When seedlings were grown on high concentrations of peptide (10 µm), root growth was suppressed; however, when seedlings were grown on low concentrations of peptide (0.1 µm), root growth was stimulated. Together, these findings indicate that AtCLEs1-7 may be the target peptides mimicked by HsCLEs to promote parasitism.
Asunto(s)
Arabidopsis/parasitología , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Imitación Molecular , Péptidos/metabolismo , Tylenchoidea/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas del Helminto/química , Interacciones Huésped-Parásitos/efectos de los fármacos , Ligandos , Imitación Molecular/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Filogenia , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Plantones/efectos de los fármacos , Plantones/parasitología , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ESR (CLE)-like effector proteins. These proteins have been shown to act as ligand mimics of plant CLE peptides and are required for successful nematode infection; however, the receptors for nematode CLE-like peptides have not been identified. Here we demonstrate that CLV2 and CORYNE (CRN), members of the receptor kinase family, are required for nematode CLE signaling. Exogenous peptide assays and overexpression of nematode CLEs in Arabidopsis demonstrated that CLV2 and CRN are required for perception of nematode CLEs. In addition, promoter-reporter assays showed that both receptors are expressed in nematode-induced syncytia. Lastly, infection assays with receptor mutants revealed a decrease in both nematode infection and syncytium size. Taken together, our results indicate that perception of nematode CLEs by CLV2 and CRN is not only required for successful nematode infection but is also involved in the formation and/or maintenance of nematode-induced syncytia.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de la Membrana/metabolismo , Nematodos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Nematodos/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/parasitología , Regiones Promotoras Genéticas/genética , Unión Proteica , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Transducción de SeñalRESUMEN
Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.
Asunto(s)
Arabidopsis/parasitología , Pared Celular/parasitología , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/parasitología , Tylenchoidea/metabolismo , Animales , Arabidopsis/metabolismo , Pared Celular/metabolismo , Femenino , Nematodos , Raíces de Plantas/metabolismo , Raíces de Plantas/parasitología , Transporte de ProteínasRESUMEN
Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.