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In October 2018, symptoms of leaf necrosis, wilted shoots, and stunted growth were observed on the upper portion of the 7-month-old cannabis (Cannabis sativa L.) plants in Mendocino County, California, U.S.A. Foliar symptoms were followed by a rapid death of the plants within 24 hours. Out of 200 affected plants, 80% (160/200) were symptomatic. All affected plants were grown in non-woven polypropylene containers (Smart pots, Oklahoma, USA) set directly on the ground approximately 3 m apart outdoors, surrounded by native forest (Quercus spp., Pseudotsuga menzeisii). Closer examination of the C. sativa plants revealed diagnostic signs of Armillaria root disease: white mycelial fans at the base of the woody stem (root collar) and abundant rhizomorphs on the roots and root collar (Supplementary Fig 1A). Also, both woody roots and the root collar exhibited severely rotted wood. Rotted wood, mycelial fans, and rhizomorphs (n=20) were surface sterilized with 0.6% sodium hypochlorite, rinsed with sterile water, and plated on PDA amended with tetracycline (1 mg/L). Sixteen cultures with morphological characters of Armillaria sp. (regular colony margin, no spore structures, no clamp connections) were recovered (Baumgartner et al., 2011). Species identity was confirmed by sequencing the internal transcribed spacer (ITS) region of rDNA and the translation elongation factor subunit 1-alpha (TEF1a) loci (White et al. 1990, Baumgartner et al 2010). Sequences (GenBank nos. MT248417 and MT259788) were compared with those in the NCBI GenBank database using a BLAST search, revealing 876/881bp matching with Armillaria gallica ITS sequence, GenBank no KP960553, and 146/150bp matching with TEF1a sequence from a North America A. gallica isolate, GenBank no. JF895844 (Brazee et al., 2011). Pathogenicity tests were conducted twice using two A. gallica isolates (15389-1 and 15389-2) by inoculating sterile, 1-month old, rooted tissue-cultured cannabis plants of 'Wedding Cake' with 7.5 ml of homogenized A. gallica liquid inoculum (Baumgartner et al., 2010), added aseptically to the surface of the vermiculite, near the plant stem (Ford et al., 2017). Eight plants were inoculated and two (using sterile water instead of inoculum) were used as negative controls. Plants were incubated at 21-26 °C under 40 to 80 µmol·m-2·s-1 from full spectrum light source with an 18/6 photoperiod to support vegetative growth. Plants were watered with 25 ml sterile nutrient solution (Cutting Edge Solutions, Santa Rosa, CA, U.S.A.) at 1 to 2-week intervals, according to the plant's need. At eight weeks post inoculation, all eight inoculated plants showed symptoms of yellowing and wilting. Uptake of the nutrient solution and water had also stopped by this time. The two non-inoculated plants, however, remained healthy throughout the 8-week period (Supplementary Fig 1B). At the end of the experiment, samples were taken aseptically from the crowns and roots of each plant and plated on water agar amended with streptomycin (100 µg/ml) and benomyl (4 µg/ml). Hyphae were subcultured to 0.5X PDA to confirm species identity through ITS and TEF1a. A. gallica was reisolated from affected crowns and stems. This is the first report of A. gallica causing root and crown rot of C. sativa. Considering the expanding cultivation area of Cannabis crops due to legalization of the industry in many U.S. states, A. gallica root and crown rot may become a serious issue affecting the industry, even for plants maintained in non-woven polypropylene containers in direct contact with soil.
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The article contains raw and analyzed data related to the research article "Neuronal ceroid lipofuscinosis genes, CLN2, CLN3, CLN5 are spatially and temporally co-expressed in a developing mouse brain" (Fabritius et al., 2014) [1]. The processed data gives an understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN1, CLN2, CLN3 and CLN5 genes in a murine model. The data shows relationship between the expression pattern of these genes during neural development. Immunohistochemistry was used to identify known interneuronal markers for neurotransmission and cell proliferation: parvalbumin, somatostatin subpopulations of interneurons. Non-radioactive in-situ hybridization detected CLN5 mRNA in the hippocampus. Throughout the development strong expression of CLN genes were identified in the germinal epithelium and in ventricle regions, cortex, hippocampus, and cerebellum. This provides supportive evidence that CLN1, CLN2, CLN3 and CLN5 genes may be involved in synaptic pruning.
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Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.
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Interacciones Huésped-Parásitos , Phytophthora infestans/fisiología , ARN de Algas/metabolismo , Solanum lycopersicum/fisiología , Simbiosis , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Calor , Solanum lycopersicum/parasitología , Datos de Secuencia Molecular , Phytophthora infestans/genética , Phytophthora infestans/crecimiento & desarrollo , Enfermedades de las Plantas/parasitología , ARN de Algas/genética , Estrés FisiológicoRESUMEN
Infantile Neuronal Ceroid Lipofuscinosis (INCL) results from mutations in the palmitoyl protein thioesterase (PPT1, CLN1) gene and is characterized by dramatic death of cortical neurons. We generated Ppt1Deltaex4 mice by a targeted deletion of exon 4 of the mouse Ppt1 gene. Similar to the clinical phenotype, the homozygous mutants show loss of vision from the age of 8 weeks, seizures after 4 months and paralysis of hind limbs at the age of 5 months. Autopsy revealed a dramatic loss of brain mass and histopathology demonstrated accumulation of autofluorescent granular osmiophilic deposits (GRODS), both characteristic of INCL. At 6 months, the homozygous Ppt1Deltaex4 mice showed a prominent loss of GABAergic interneurons in several brain areas. The transcript profiles of wild-type and mutant mouse brains revealed that most prominent alterations involved parts of the immune response, implicating alterations similar to those of the aging brain and neurodegeneration. These findings make the Ppt1Deltaex4 mouse an interesting model for the inflammation-associated death of interneurons.
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Corteza Cerebral/metabolismo , Encefalitis/genética , Interneuronas/metabolismo , Degeneración Nerviosa/genética , Lipofuscinosis Ceroideas Neuronales/genética , Tioléster Hidrolasas/genética , Animales , Animales Recién Nacidos , Ceguera Cortical/genética , Ceguera Cortical/metabolismo , Ceguera Cortical/fisiopatología , Muerte Celular/genética , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Modelos Animales de Enfermedad , Encefalitis/patología , Encefalitis/fisiopatología , Femenino , Eliminación de Gen , Marcación de Gen , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/ultraestructura , Interneuronas/patología , Interneuronas/ultraestructura , Masculino , Ratones , Ratones Mutantes Neurológicos , Microscopía Electrónica de Transmisión , Mutación/genética , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Lipofuscinosis Ceroideas Neuronales/patología , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Parálisis/genética , Parálisis/metabolismo , Parálisis/fisiopatología , Fenotipo , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/fisiopatología , Vísceras/metabolismo , Vísceras/patología , Vísceras/ultraestructura , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Neuronal ceroid lipofuscinoses (NCL) comprise the most common group of childhood encephalopathies caused by mutations in eight genetic loci, CLN1-CLN8. Here, we have developed a novel mouse model for the human vLINCL (CLN5) by targeted deletion of exon 3 of the mouse Cln5 gene. The Cln5-/- mice showed loss of vision and accumulation of autofluorescent storage material in the central nervous system (CNS) and peripheral tissues without prominent brain atrophy. The ultrastructure of the storage material accurately replicated the abnormalities in human patients revealing mixture of lamellar profiles including fingerprint profiles as well as curvilinear and rectilinear bodies in electronmicroscopic analysis. Prominent loss of a subset of GABAergic interneurons in several brain areas was seen in the Cln5-/- mice. Transcript profiling of the brains of the Cln5-/- mice revealed altered expression in several genes involved in neurodegeneration, as well as in defense and immune response, typical of age-associated changes in the CNS. Downregulation of structural components of myelin was detected and this agrees well with the hypomyelination seen in the human vLINCL patients. In general, the progressive pathology of the Cln5-/- brain mimics the symptoms of the corresponding neurodegenerative disorder in man. Since the Cln5-/- mice do not exhibit significant brain atrophy, these mice could serve as models for studies on molecular processes associated with advanced aging.
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Envejecimiento , Encéfalo/patología , Modelos Animales de Enfermedad , Proteínas de la Membrana/fisiología , Lipofuscinosis Ceroideas Neuronales/genética , Animales , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/fisiopatología , Cartilla de ADN , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Proteínas de Membrana de los Lisosomas , Lisosomas/enzimología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Neuronal ceroid lipofuscinoses (NCLs) are recessively inherited neurodegenerative lysosomal storage disorders characterized by progressive motor and mental retardation, visual failure, and epileptic seizures. Finnish variant late infantile NCL (vLINCL(Fin)) is caused by mutations in the CLN5 gene. We have isolated the mouse Cln5 gene and analyzed its spatiotemporal expression in the central nervous system (CNS) by in situ hybridization and immunohistochemistry. Cln5 was expressed throughout the embryonic brain already at E15 and the expression steadily increased during development. Prominent expression was observed in cerebellar Purkinje cells, cerebral neurons, hippocampal pyramidal cells, and hippocampal interneurons. The expression pattern correlated with those CNS regions that get degenerated in CLN5 patients. In vitro expression of Cln5 in COS-1, HeLa, and neuronal cells further implied that mouse Cln5 is a soluble lysosomal glycoprotein, closely resembling human CLN5.
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Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Lisosomas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas de Membrana de los Lisosomas , Lisosomas/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Five members of an elicitor-like gene family from Phytophthora infestans were examined. The family was identified through the analysis of M81, a mating-induced gene. The predicted M81 product resembled a 42-kDa P. sojae glycoprotein known to elicit defense reactions in plants, including a host of P. infestans, potato. M81 was the most structurally and functionally divergent of the P. infestans genes compared with the P. sojae sequence. M81 lacked elicitor activity, had the lowest protein identity (47%), displayed mating-specific transcription, and had a novel C-terminal domain. The latter contained a 30-residue proline- and threonine-rich motif, which, remarkably, was tandemly repeated 24 to 36 times in different alleles. M81C, M81D, and M81E better resembled the P. sojae protein based on amino acid identity (63 to 75%) and conserved elicitor activity. M81C and M81D mRNA accumulated only during zoosporogenesis, while M81E expression was restricted to hyphae. M81B, an apparent pseudogene, was physically linked to M81. The protein products of each gene were predicted to be extracellular transglutaminases ranging in size from 436 to 1,607 amino acids. Genes with an elicitor, proline- and threonine-rich repeat, and both elicitor and repeat domains were widely distributed throughout Phytophthora infestans. These findings help explain the natural functions of elicitors in pathogen biology and plant-microbe interactions.
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Proteínas Algáceas/química , Phytophthora/patogenicidad , Proteínas Algáceas/genética , Alelos , Secuencia de Aminoácidos , Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes , Phytophthora/química , Phytophthora/genética , Homología de Secuencia de Aminoácido , Solanum tuberosum/microbiologíaRESUMEN
Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40- to >100-fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60- to >100-fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA-binding proteins and RNase H. The products of two other mating-induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating-induced gene was also expressed in a self-fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self-fertile heterokaryon. Besides the mating-induced genes, two extrachromosomal RNA elements were identified.