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Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process.
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Complejo III de Transporte de Electrones , Proteínas del Helminto , Proteínas Hierro-Azufre , Inmunidad de la Planta , Plastidios , Especies Reactivas de Oxígeno , Animales , Arabidopsis/parasitología , Arabidopsis/inmunología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Mutación/genética , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/inmunología , Plastidios/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Tylenchoidea/genética , Tylenchoidea/patogenicidadRESUMEN
The Meloidogyne enterolobii effector MeTCTP is a member of the translationally controlled tumour protein (TCTP) family, involved in M. enterolobii parasitism. In this study, we found that MeTCTP forms homodimers and, in this form, binds calcium ions (Ca2+ ). At the same time, Ca2+ could induce homodimerization of MeTCTP. We further identified that MeTCTP inhibits the increase of cytosolic free Ca2+ concentration ([Ca2+ ]cyt ) in plant cells and suppresses plant immune responses. This includes suppression of reactive oxygen species burst and cell necrosis, further promoting M. enterolobii parasitism. Our results have elucidated that the effector MeTCTP can directly target Ca2+ by its homodimeric form and prevent [Ca2+ ]cyt rise in plant roots, revealing a novel mechanism utilized by plant pathogens to suppress plant immunity.
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Enfermedades de las Plantas , Tylenchoidea , Animales , Citosol , Inmunidad de la Planta , Raíces de PlantasRESUMEN
Various cells within the adrenal microenvironment are important in maintaining the body homeostasis. However, our understanding of adrenal disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ's multiple homeostatic functions. We report a cellular landscape of the human adrenal gland using single-cell RNA sequencing. We reveal characteristic features of cell types within the human adrenal microenvironment and found immune activation of nonimmune cells in the adrenal endothelial cells. We also reveal that abundant immune cells occupied a lot of space in adrenal gland. Additionally, Sex-related diversity in the adrenocortical cells and different gene expression profiles between the left and right adrenal gland are also observed at single-cell resolution. Together, at single-cell resolution, the transcriptomic map presents a comprehensive view of the human adrenal gland, which serves as a fundamental baseline description of this organ and paves a way for the further studies of adrenal diseases.
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Glándulas Suprarrenales/metabolismo , Microambiente Celular , Análisis de la Célula Individual , Transcriptoma , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/inmunología , Anciano , Ritmo Circadiano , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , RNA-Seq , Factores SexualesRESUMEN
Recent studies have reported that plant-parasitic nematodes facilitate their infection by suppressing plant immunity via effectors, but the inhibitory mechanisms remain poorly understood. This study found that a novel effector MgMO289 is exclusively expressed in the dorsal esophageal gland of Meloidogyne graminicola and is up-regulated at parasitic third-/fourth-stage juveniles. In planta silencing of MgMO289 substantially increased plant resistance to M. graminicola. Moreover, we found that MgMO289 interacts with a new rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04), and that rice cytosolic COPPER/ZINC -SUPEROXIDE DISMUTASE 2 (cCu/Zn-SOD2) is the target of OsHPP04. Rice plants overexpressing OsHPP04 or MgMO289 exhibited an increased susceptibility to M. graminicola and a higher Cu/Zn-SOD activity, but lower O2â¢- content, when compared with wild-type plants. Meanwhile, immune response assays showed that MgMO289 could suppress host innate immunity. These findings reveal a novel pathway for a plant pathogen effector that utilizes the host O2â¢--scavenging system to eliminate O2â¢- and suppress plant immunity.
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Oryza , Tylenchoidea , Animales , Cobre , Metalochaperonas , Oryza/genética , Enfermedades de las PlantasRESUMEN
BACKGROUND: Prostate cancer (PCa) is still a serious male malignant disease across the world. However, no exact pathogenesis had been explained. Although adenylosuccinate lyase (ADSL) gene was identified to be important in PCa early in 1987, its comprehensive functions for PCa have not been presented. METHODS: The cBioPortal for Cancer Genomics, Oncomine and GEO database were retrieved to investigate the associations between of the ADSL gene and PCa. Then, the PC-3, DU145 and C4-2B cell lines were applied in vitro experiments. RNA sequencing and further western blot (WB) were applied to explore the potential mechanisms of ADSL gene in PCa. RESULTS: Based on PCa clinical datasets, we firstly found ADSL gene highly expressed in PCa tissues. Moreover, its transcript level increased in the metastatic PCa further. Elevated ADSL gene expression indicated a poor prognosis of PCa. While inhibiting the expression of ADSL with siRNA, the ability of cell proliferation and migration all declined markedly, with increased cell apoptosis inversely. Most of cells were blocked in the G0/G1 phase. Additionally, RNA sequencing also discovered the inactivity of cell cycle pathway after ADSL knockdown, which had also confirmed on the proteins levels. CONCLUSIONS: Our study identified the ADSL as an oncogene of PCa through regulating the cell cycle pathway firstly, with explicit cell and clinical phenotypes. Further mechanisms were needed to confirm its carcinogenic effect.
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An emerging threat to agriculture, Meloidogyne enterolobii Yang & Eisenback, 1983, is a tropical species and considered to be the most damaging root-knot nematode (RKN) in the world because of its wide host range, aggressiveness, and ability to overcome resistance to RKN in many crops. It was first detected in the United States on ornamental plants in Florida in 2001 but has since been identified in North Carolina, South Carolina, and Louisiana. Several thousand RKN populations were collected from North Carolina field crops, ornamental plants, and turfgrasses for species identification in the Nematode Assay Laboratory in the North Carolina Department of Agriculture & Consumer Services. From 2006 to 2019, root systems showing galling symptoms were dissected under the microscope, and females were obtained for DNA analysis. When only soil samples were submitted, the second-stage juveniles or males were used instead. Molecular characterization was performed via polymerase chain reaction with species-specific primers and DNA sequencing on the ribosomal DNA 18S-ITS1-5.8S and 28S D2/D3 and mitochondrial DNA CoxII-16S. One hundred thirty-five representative RKN populations from North Carolina were characterized and identified as M. enterolobii. Six populations from China where the species was originally described were included in this study for identity confirmation and comparison. As of December 2019, M. enterolobii has been confirmed from a limited number of fields in 11 North Carolina counties: Columbus, Craven, Greene, Harnett, Johnston, Lenoir, Nash, Pitt, Sampson, Wayne, and Wilson. Currently, M. enterolobii is the most important emerging RKN species in the United States and causes severe damage to agronomic and horticultural crops, especially sweetpotato in North Carolina.
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Tylenchoidea , Animales , China , Florida , Louisiana , North Carolina , Enfermedades de las Plantas , Raíces de Plantas , South Carolina , Tylenchoidea/genéticaRESUMEN
Basilaphelenchus brevistylus n. sp. was isolated from masson pine (Pinus massoniana) in Guangdong province, China. The new species is characterized by an offset lip region, short stylet (female stylet 4.5-5.5 µm and male stylet 4-5 µm long) with three elongate posteriorly directed knobs, posteriorly located metacorpal valve and lateral field composed of three lines. The female has an elongate postuterine sac and a short conical tail, uniformly narrowing toward a sharp tip, or tapering to a slightly offset mucronate tip in a few individuals. The male has a conical tail with a sharp terminal mucro, three pairs of caudal papillae, and small arcuate spicules with a bluntly rounded condylus and small pointed rostrum. B. brevistylus n. sp. can be distinguished from all described Basilaphelenchus nematodes by numerous morphological and morphometrical traits, especially the tail morphology of both sexes and stylet length. In addition, molecular phylogenetic trees inferred from rRNA small subunit and D2-D3 expansion domains of large subunit revealed that this nematode belongs to the Basilaphelenchus, and is clearly different from all the other Basilaphelsenchus species.
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BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.
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Análisis de la Célula Individual , Transcriptoma , Vejiga Urinaria/citología , Vejiga Urinaria/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/análisis , Receptores Histamínicos H2/análisis , Análisis de Secuencia de ARN , Troponina T/análisisRESUMEN
Bursaphelenchus yongensis was first reported in China, and later found in Japan and Korea. It is characterized by a relatively slim body (a = 42 and 57 for females and males, respectively). The excretory pore is located at level of median bulb, the lateral field has three lines, and a small vulval flap is present. A long post-uterine branch extends 2/3 to 3/4 of the vulva to anus distance. The conoid female tail has a 2-5 µm long mucron in the central position at the terminus. Spicules are small, condylus high and strongly dorsally bent. Subsequently Bursaphelenchus uncispicularis was described from China. Both morphological characters and morphometrics are very similar to B. yongensis, except for the number of lateral lines (4 vs 3) and male caudal papillae (7 vs 4). Re-examination of type material and a Beijing population of B. yongensis determined that B. yongensis has 7 caudal papillae instead of 4 as originally reported. It is possible that the poor condition of the type specimens of B. uncispicularis could have created difficulty in the determination of lateral line number. Unfortunately, type material of B. uncispicularis has been lost. Therefore, there is no evidence that B. uncispicularis exists. It is now established that B. yongensis is present in China, Japan and Korea with a common host species (P. thunbergii) and a common widespread vector (Cryphalus fulvus). Therefore, based on the geographic, ecological, molecular, and morphological data, we propose Bursaphelenchus uncispicularis Zhuo, Li, Li, Yu & Liao, 2007 as a junior synonym of B. yongensis Gu, Braasch, Burgermeister, Brandstetter & Zhang, 2006.
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Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.
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Proteínas del Helminto/metabolismo , Oryza/parasitología , Enfermedades de las Plantas/parasitología , Plantas Modificadas Genéticamente/parasitología , Tylenchoidea/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Glicosilación , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Oryza/genética , Oryza/inmunología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/metabolismo , Transporte de Proteínas , Proteolisis , Tylenchoidea/genética , Tylenchoidea/inmunologíaRESUMEN
BACKGROUND The present study aimed to evaluate the difference in microbiota diversity in the oral cavity and fluid bronchoalveolar lavage (BALF) of patients with lung cancer. MATERIAL AND METHODS Buccal (saliva) and lower respiratory tract BALF samples were collected from 51 patients with primary bronchogenic carcinoma and 15 healthy controls, and bacterial genomic DNA was extracted. High-throughput 16S rDNA amplicon sequencing was performed, and microbial diversity, composition, and functions of microbiota were analyzed by bioinformatics methods. RESULTS Patients with lung cancer have lower microbial diversity than healthy controls in both saliva and BALF samples. Significant segregation was observed between the different pathological types of lung cancer groups and the control group regardless of the sampling site. Treponema and Filifactor were identified as potential bacterial biomarkers in BALF samples, while Filifactor was ideal to distinguish healthy controls from lung cancer patients. Moreover, the predictive variation analysis of the KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway showed that the metabolic differences in microbiota varied by sampling site. CONCLUSIONS Lung cancer patients carry a different and less diverse microorganism community than healthy controls. Certain bacterial taxa might be associated with lung cancer, but the exact species depends on the sampling site and the pathological type. This study provides basic data on the microbiota diversity in BALF and saliva samples from lung cancer patients. Further investigation with a larger sample size should help validate the enriched species in different pathological types of lung cancers.
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Líquido del Lavado Bronquioalveolar/microbiología , Carcinoma Broncogénico/microbiología , Neoplasias Pulmonares/microbiología , Saliva/microbiología , Adulto , Anciano , Bacterias/genética , Bacterias/aislamiento & purificación , Biomarcadores de Tumor/análisis , Lavado Broncoalveolar/métodos , Femenino , Humanos , Masculino , Microbiota , Persona de Mediana Edad , Datos PreliminaresRESUMEN
BACKGROUND: Previous studies have revealed that the single nucleotide polymorphism (SNP) rs2188380 was identified as a novel locus of gout. Interestingly, gout resulting from high serum uric acid (SUA) was also identified to be associated with the risk of breast cancer (BC). We hypothesized that maybe there was a relationship between rs2188380 and the risk of BC. Therefore, our study was conducted to investigate whether this novel gout-related SNP (rs2188380) was associated with BC risk as well as the clinical and pathological characteristics in the southwest Chinese population. MATERIALS AND METHODS: We performed a case-control study including 104 breast cancer patients and 112 healthy controls to investigate whether rs2188380 is associated with BC risk in the southwest Chinese population. The genotyping was performed by the SNP scan method. General characteristics and clinicopathological characteristics of tumors were also included in the analysis. The statistical evaluations were performed using the Student t test, the chi-square test or Fisher's exact test, and unconditional logistic regression analysis. RESULTS: The C/C genotype of rs2188380 might be related to BC risk to some extent compared with G/G genotype (OR = 9.241, 95% CI = 1.122-76.101, P = 0.039). Furthermore, after adjusting the age, the association still existed (OR = 8.788, 95% CI = 1.063-72.636, P = 0.044). However, no significant association was observed between rs2188380 and the clinicopathological characteristics of BC. CONCLUSIONS: Our study primarily indicated that rs2188380 might have a potential association with BC risk to some extent. With a limited sample size and statistical power, further studies based on larger populations are needed to confirm the association.
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Neoplasias de la Mama/genética , ADN Intergénico , Polimorfismo de Nucleótido Simple , Adulto , Pueblo Asiatico/genética , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana EdadRESUMEN
Evidence is emerging that plant-parasitic nematodes can secrete effectors to interfere with the host immune response, but it remains unknown how these effectors can conquer host immune responses. Here, we depict a novel effector, MjTTL5, that could suppress plant immune response. Immunolocalization and transcriptional analyses showed that MjTTL5 is expressed specifically within the subventral gland of Meloidogyne javanica and up-regulated in the early parasitic stage of the nematode. Transgenic Arabidopsis lines expressing MjTTL5 were significantly more susceptible to M. javanica infection than wild-type plants, and vice versa, in planta silencing of MjTTL5 substantially increased plant resistance to M. javanica. Yeast two-hybrid, coimmunoprecipitation and bimolecular fluorescent complementation assays showed that MjTTL5 interacts specifically with Arabidopsis ferredoxin : thioredoxin reductase catalytic subunit (AtFTRc), a key component of host antioxidant system. The expression of AtFTRc is induced by the infection of M. javanica. Interaction between AtFTRc and MjTTL could drastically increase host reactive oxygen species-scavenging activity, and result in suppression of plant basal defenses and attenuation of host resistance to the nematode infection. Our results demonstrate that the host ferredoxin : thioredoxin system can be exploited cunningly by M. javanica, revealing a novel mechanism utilized by plant-parasitic nematodes to subjugate plant innate immunity and thereby promoting parasitism.
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Arabidopsis/parasitología , Depuradores de Radicales Libres/metabolismo , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Inmunidad de la Planta , Especies Reactivas de Oxígeno/metabolismo , Tylenchoidea/fisiología , Animales , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Teorema de Bayes , Clonación Molecular , Simulación por Computador , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Genes de Helminto , Genes de Plantas , Proteínas del Helminto/química , Proteínas del Helminto/genética , Peróxido de Hidrógeno/metabolismo , Mutación/genética , Parásitos , Moléculas de Patrón Molecular Asociado a Patógenos , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Unión Proteica , Dominios Proteicos , Interferencia de ARN , Tylenchoidea/genética , Regulación hacia ArribaRESUMEN
Tylenchulus semipenetrans is an economically important plant-parasitic nematode occurring in all citrus-producing regions of the world and causing a disease called "slow decline". For the rapid detection of this nematode, a loop-mediated isothermal amplification (LAMP) assay was developed, based on the ribosomal DNA internal transcribed spacer sequence. The optimal condition for the LAMP assay was 65°C for 50 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR) and restriction analysis with the BamHI enzyme, and by adding SYBR Green I to the LAMP products for visual inspection. The LAMP assay was highly specific for the detection of T. semipenetrans populations from different geographical origins. It was also sensitive, detecting a tenth of the DNA from an individual specimen of T. semipenetrans, which was 10 times more sensitive than conventional PCR. The LAMP protocol was applied to natural citrus rhizosphere soil samples from several orchards in China and the results were fast, sensitive, robust, and accurate. This study is the first to provide a diagnostic tool for T. semipenetrans using DNA extracted directly from citrus rhizosphere soils. This LAMP assay could be used as a practical molecular tool to identify T. semipenetrans and diagnose slow decline disease, even in remote locations.
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Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen.
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Agrobacterium tumefaciens/crecimiento & desarrollo , Agrobacterium tumefaciens/genética , Técnicas de Transferencia de Gen , Selección Genética , Ustilago/crecimiento & desarrollo , Ustilago/genética , Transformación GenéticaRESUMEN
Meloidogyne javanica is one of the most widespread and economically important sedentary endoparasites. In this study, a comparative transcriptome analysis of M. javanica between pre-parasitic second-stage juveniles (Pre-J2) and parasitic juveniles (Par-J3/J4) was conducted. A total of 48,698 unigenes were obtained, of which 18,826 genes showed significant differences in expression (p < 0.05). In the differentially expressed genes (DEGs) from transcriptome data at Par-J3/J4 and Pre-J2, a large number of unigenes were annotated to the C-type lectin (CTL, Mg01965), the cathepsin L-like protease (Mi-cpl-1), the venom allergen-like protein (Mi-mps-1), Map-1 and the cellulase (endo-ß-1,4-glucanase). Among seven types of lectins found in the DEGs, there were 10 CTLs. The regulatory roles of Mj-CTL-1, Mj-CTL-2 and Mj-CTL-3 in plant immune responses involved in the parasitism of M. javanica were investigated. The results revealed that Mj-CTL-2 could suppress programmed cell death (PCD) triggered by Gpa2/RBP-1 and inhibit the flg22-stimulated ROS burst. In situ hybridization and developmental expression analyses showed that Mj-CTL-2 was specifically expressed in the subventral gland of M. javanica, and its expression was up-regulated at Pre-J2 of the nematode. In addition, in planta silencing of Mj-CTL-2 substantially increased the plant resistance to M. javanica. Moreover, yeast co-transformation and bimolecular fluorescence complementation assay showed that Mj-CTL-2 specifically interacted with the Solanum lycopersicum catalase, SlCAT2. It was demonstrated that M. javanica could suppress the innate immunity of plants through the peroxide system, thereby promoting parasitism.
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The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.
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Secretory effector proteins expressed within the esophageal glands of root-knot nematodes (Meloidogyne spp.) are thought to play key roles in nematode invasion of host roots and in formation of feeding sites necessary for nematodes to complete their life cycle. In this study, a novel effector protein gene designated as Mj-nulg1a, which is expressed specifically within the dorsal gland of Meloidogyne javanica, was isolated through suppression subtractive hybridization. Southern blotting and BLAST search analyses showed that Mj-nulg1a is unique for Meloidogyne spp. A real-time reverse-transcriptase polymerase chain reaction assay showed that expression of Mj-nulg1a was upregulated in parasitic second-stage juveniles and declined in later parasitic stages. MJ-NULG1a contains two putative nuclear localization signals and, consistently, in planta immunolocalization analysis showed that MJ-NULG1a was localized in the nuclei of giant cells during nematode parasitism. In planta RNA interference targeting Mj-nulg1a suppressed the expression of Mj-nulg1a in nematodes and attenuated parasitism ability of M. javanica. In contrast, transgenic Arabidopsis expressing Mj-nulg1a became more susceptible to M. javanica infection than wild-type control plants. These results depict a novel nematode effector that is targeted to giant cell nuclei and plays a critical role in M. javanica parasitism.
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Arabidopsis/parasitología , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Helminto/genética , Enfermedades de las Plantas/parasitología , Tylenchoidea/genética , Animales , Anticuerpos Antihelmínticos , Secuencia de Bases , Núcleo Celular/metabolismo , ADN de Helmintos/química , ADN de Helmintos/genética , Susceptibilidad a Enfermedades , Células Gigantes/metabolismo , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Solanum lycopersicum/citología , Solanum lycopersicum/parasitología , Datos de Secuencia Molecular , Señales de Localización Nuclear , Raíces de Plantas/parasitología , Plantas Modificadas Genéticamente , Interferencia de ARN , ARN de Helminto/genética , Análisis de Secuencia de ADN , Tylenchoidea/crecimiento & desarrollo , Tylenchoidea/fisiología , Tylenchoidea/ultraestructura , Regulación hacia ArribaRESUMEN
This study describes the molecular and biochemical characterization of the ß-1,4-endoglucanase gene (Mj-eng-3) from the root knot nematode Meloidogyne javanica. A 2156-bp genomic DNA sequence of Mj-eng-3 containing six introns was obtained. Mj-eng-3 was localized in the subventral esophageal glands of M. javanica juveniles by in situ hybridization. Real-time RT-PCR assay showed that the highest transcriptional level of Mj-eng-3 occurred in pre-parasitic second-stage juveniles, and this high expression persisted in parasitic second-stage juveniles. Recombinant MJ-ENG-3 degraded carboxymethylcellulose and optimum enzyme activity at 40°C and pH 8.0. EDTA, Mg(2+), Mn(2+), Ca(2+), Co(2+), and Cu(2+) did not affect the activity of MJ-ENG-3; however, Zn(2+) and Fe(2+) inhibited MJ-ENG-3 enzyme activity. In planta Mj-eng-3 RNAi assay displayed a reduction in the number of nematodes and galls in transgenic tobacco roots. These results suggested that MJ-ENG-3 could be secreted by M. javanica to degrade the cellulose of plant cell walls to facilitate its entry and migration during the early stages of parasitism.
Asunto(s)
Celulasa/genética , Celulasa/metabolismo , Nicotiana/parasitología , Tylenchoidea/enzimología , Tylenchoidea/genética , Animales , ADN Complementario/química , ADN de Helmintos/química , Exones , Regulación del Desarrollo de la Expresión Génica , Concentración de Iones de Hidrógeno , Hibridación in Situ , Intrones , Enfermedades de las Plantas/parasitología , Raíces de Plantas/parasitología , Interferencia de ARN , ARN de Helminto/genética , Alineación de Secuencia , Temperatura , Tylenchoidea/crecimiento & desarrolloRESUMEN
Heteroderafengi n. sp. is described and illustrated from bamboo (Phyllostachys pubescens Mazel) based on morphology and molecular analyses of rRNA LSU D2D3 region and ITS. This new species belongs to the Cyperi group. Cysts are characterized by prominent vulval cone with ambifenestrate, bifurcate underbridge that is thicker in middle and a 47.0 (40.0-60.0) µm long vulval slit, but without bullae. The second-stage juveniles are characterized by a 23.2 (22.0-24.0) µm long stylet with slightly projected or anteriorly flattened knobs, three incisures in lateral field, a 70.2 (62.5-77.0) µm long tail with bluntly rounded terminus and hyaline portion ca 58.9 (50.0-62.5)% of the tail length. Males are characterized by a 25.1 (24.5-26.3) µm long stylet with rounded knobs sloping posteriorly, four incisures in lateral field, a 29.8 (27.5-31.3) µm long spicule with bifurcate tip. Phylogenetic analysis shows that the species has unique D2D3 and ITS rRNA sequences and RFLP-ITS-rRNA profiles. Heteroderafengi n. sp. is closest to H. elachista in dendrograms inferred from both DNA sequences.