Detección, transmisión y caracterización del fitoplasma asociado a la enfermedad del decaimiento del peral

Consultable des del TDXTítol obtingut de la portada digitalitzadaSe ha realizado una prospección en 1500 parcelas de cultivo de peral, para evaluar la extensión de la enfermedad del Decaimiento del peral (PD) en el Nordeste de España. Un 7% de las parcelas observadas presentaron síntomas de la enfermedad. Paralelamente, se ha evaluado la incidencia de la enfermedad en 45 de estas parcelas, por inspección visual de 500 árboles, en cada una de ellas. La presencia del fitoplasma fue confirmada mediante nested-PCR. Finalmente, la caracterización del fitoplasma, indicó que la enfermedad estaba causada por un único fitoplasma, el fitoplasma de PD. Se ha evaluado la expresión de síntomas de la enfermedad de PD a lo largo del año, en tres variedades distintas. Cada variedad presentó unos síntomas asociados característicos, hecho que puede ser de utilidad en nuevas prospecciones. La presencia del fitoplasma se ha examinado mediante la técnica de nested-PCR en un total de 43 árboles. En los tres casos, la máxima detección del fitoplasma fue alcanzada en Diciembre. Paralelamente, en este estudio se ha analizado qué parte de la planta (nervios, yemas o tallo) era la más adecuada para realizar una detección del fitoplasma mediante PCR. Se obtuvo la detección más fiable en tallo. Finalmente, se ha examinado el estado funcional del fitoplasma de PD durante el invierno, mediante transmisión del fitoplasma por injerto a perales sanos. Los resultados demostraron que el fitoplasma puede permanecer en estado funcional en la parte aérea del árbol durante la fase de hibernación. Se ha estudiado el papel de Cacopsylla pyri (Homoptera; psyllidae) en la transmisión del fitoplasma del Decaimiento del peral (PD) en el Nordeste de España. Los resultados obtenidos demostraron que C. pyri transmite el fitoplasma a peral y medios artificiales, y por tanto es, como en otros países del área mediterránea, vector de la enfermedad en España. Los porcentajes de psilas infectadas fueron similares entre sexos, sin embargo las hembras transmitieron el fitoplasma en mayor porcentaje que los machos. Por último, se ha puesto a punto una técnica para separar el ADN del genoma de los fitoplasmas, del de las plantas que los hospedan, gracias al alto contenido en A+T de los fitoplasmas en comparación con el de las plantas huésped. Los resultados muestran que un 90% de los clones recombinantes contenían ADN de fitoplasma. Por secuenciación y comparación con el banco de datos de NCBI fue confirmado el origen bacteriano de las secuencias, Finalmente mediante el diseño de cebadores para alguna de estas secuencias y posterior análisis por PCR se corroboró que los fragmentos de ADN clonados eran fitoplasmáticos. La técnica de dot.blot ha sido utilizada para detectar el fitoplasma del Decaimiento del peral (PD) con secuencias de ADN no ribosómicas. Para este propósito, se escogieron 2 fragmentos obtenidos a partir de la técnica anterior y fueron marcados con digoxigenina y empleados como sondas, para hibridar con extractos de ADN de perales y psilas infectados con este microorganismo. El fitoplasma de PD se detectó en ambos extractos. Se han realizado también ensayos para evaluar la sensibilidad de estas sondas en la detección del fitoplasma de PD. Para ello, se analizaron extractos de ADN de psilas, por nested-PCR e hibridación por dot.blot. Los resultados obtenidos fueron similares con ambas técnicas. Por ello, se propone la hibridación ADN / ADN como una alternativa a la PCR, Por último, el ADN de diferentes fitoplasmas, se ha utilizado para determinar si los cebadores diseñados para estos dos fragmentos eran específicos para el fitoplasma de PD, o amplificaban secuencias de otros fitoplasmas. Al obtenerse una banda específica con ADN del fitoplasma de la Proliferación del manzano (AP) y con el del PD, se confirma la relación filogenética existente entre ambos.A total area of 1,500 Ha of commercial plots was surveyed to study the extent of pear decline disease and its relative importance in northeastern Spain. A preliminary evaluation indicated that around 7% of the plots had symptoms of the disease. At the same time, pear decline incidence (PDI) was evaluated in 45 plots. The presence of pear decline (PD) phytoplasma in these plots was confirmed by PCR amplification of phytoplasma DNA with universal or group-specific primers. Restriction fragment length polymorphism (RFLP) analyses also showed the presence of a unique phytoplasma strain. The symptom expression of PD disease in different cultivars was evaluated throughout the year. The relationship between the presence of symptoms and detection of PD by PCR in these cultivars was also studied. Results showed that the nested PCR, using specific primers to detect the DNA from PD phytoplasma, is the most accurate method to identify the total percentage of affected trees. Seasonal detection of pear decline phytoplasma was studied in 43 infected trees. The presence of the phytoplasma waa analysed by nested PCR. The three cultivars showed different pattern of detection. The maximum detection rate of pear decline phytoplasma occurred in December in the three orchards. At the same time, leaf midribs, buds and stems were compared to determine which sample was more reliable for phytoplasma detection. The best indicators were stems. The presence of phytoplasma in sieve tubes during the dormant season was determined by grafting. The results suggest that phytoplasmas could overwinter in shoots, with the implication that vegetative propagation during this period could also disseminate the disease. The frequency of Pear decline-positive insects and transmission of Pear decline (PD) phytoplasma by Cacopsylla pyri has been studied for the first time in Spain. Results indicate that the frequency of PD positive psyllids changes through the year and that C. pyri transmits the Pear decline associated disease agent in Spain. Phytoplasma transmission was also effective under laboratory conditions using a feeding medium. Although the percentage of PD positive psyllids was similar in both genders, PD phytoplasma transmission by females was significantly higher than by males. A method was developed for genome analysis of phytoplasmas, bacterial plant pathogens that cannot yet be cultivated in vitro, taking into account the different A/T content in the DNA of the pathogen and its plant host. Results showed that about 90% of recombinant clones appeared to harbour phytoplasma specific DNA inserts. Sequencing of randomly selected clones was carried out, and comparison with the NCBI database confirmed the phytoplasmatic origin. Some sequences could also be assigned a putative function. The origin of the recombinant clones was further confirmed by the generation of specific amplicons from the phytoplasma infected and not healthy plant, using PCR primers devised from the sequences of the recombinant clones. A non-isotopic hybridisation procedure was used to detect pear decline (PD) phytoplasma. Two new digoxigenin labelled DNA probes obtained from putative thymidilate kinase and peptide release genes of the PD phytoplasma were used. Hybridisations of these non-ribosomal probes with dot-blotted total nucleic acid extracts on nylon membranes allowed the detection of PD phytoplasma in infected plants and individual Cacopsylla pyri vectors. Assays to compare dot-blot hybridisation and nested-PCR procedures showed that the dot-blot hybridisation is a more reliable assay than nested-PCR. We propose the use of dot-blot hybridisation as a suitable technique for specific PD detection in epidemiological studies where there are a large number of samples to be tested

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

Background: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. - Results: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. - Conclusion: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants

Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre in which vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research

Description of Genetic Variants in BRCA Genes in Mexican Patients with Ovarian Cancer: A First Step towards Implementing Personalized Medicine

Abstract Gynecologic cancers are among the leading causes of death worldwide, ovarian cancer being the one with the highest mortality rate. Olaparib is a targeted therapy used in patients presenting mutations in BRCA1 and BRCA2 genes. The aim of this study was to describe BRCA1 and BRCA2 gene variants in Mexican patients with ovarian cancer. Sequencing of BRCA1 and BRCA2 genes from tumors of 50 Mexican patients with ovarian cancer was made in a retrospective, non-randomized, and exploratory study. We found genetic variants in 48 of 50 cases. A total of 76 polymorphic variants were found in BRCA1, of which 50 (66%) had not been previously reported. Furthermore, 104 polymorphic variants were found in BRCA2, of which 63 (60%) had not been reported previously. Of these polymorphisms, 5/76 (6.6%) and 4/104 (3.8%) were classified as pathogenic in BRCA1 and BRCA2, respectively. We have described the genetic variants in BRCA1 and BRCA2 of tumors from Northeast Mexican patients with sporadic ovarian cancers. Our results showed that the use of genetic testing helps recognize patients that carry pathogenic variants which could be beneficial for personalized medicine treatments. Keywords: BRCA; ovarian cancer; personalized therapy; sequencin

Towards a combined use of geophysics and remote sensing techniques for the characterization of a singular building: “El Torreón” (the tower) at Ulaca oppidum (Solosancho, Ávila, Spain)

This research focuses on the study of the ruins of a large building known as “El Torreón” (the Tower), belonging to the Ulaca oppidum (Solosancho, Province of Ávila, Spain). Different remote sensing and geophysical approaches have been used to fulfil this objective, providing a better understanding of the building’s functionality in this town, which belongs to the Late Iron Age (ca. 300–50 BCE). In this sense, the outer limits of the ruins have been identified using photogrammetry and convergent drone flights. An additional drone flight was conducted in the surrounding area to find additional data that could be used for more global interpretations. Magnetometry was used to analyze the underground bedrock structure and ground penetrating radar (GPR) was employed to evaluate the internal layout of the ruins. The combination of these digital methodologies (surface and underground) has provided a new perspective for the improved interpretation of “El Torreón” and its characteristics. Research of this type presents additional guidelines for better understanding of the role of this structure with regards to other buildings in the Ulaca oppidum. The results of these studies will additionally allow archaeologists to better plan future interventions while presenting new data that can be used for the interpretation of this archaeological complex on a larger scale

Endocytosis and early endosome motility in filamentous fungi.

types: REVIEWOpen Access funded by Biotechnology and Biological Sciences Research CouncilHyphal growth of filamentous fungi requires microtubule-based long-distance motility of early endosomes. Since the discovery of this process in Ustilago maydis, our understanding of its molecular basis and biological function has greatly advanced. Studies in U. maydis and Aspergillus nidulans reveal a complex interplay of the motor proteins kinesin-3 and dynein, which co-operate to support bi-directional motion of early endosomes. Genetic screening has shed light on the molecular mechanisms underpinning motor regulation, revealing Hook protein as general motor adapters on early endosomes. Recently, fascinating insight into unexpected roles for endosome motility has emerged. This includes septin filament formation and cellular distribution of the machinery for protein translation.BBSR

Metagenomics: DNA sequencing of environmental samples

While genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent but a fraction of the living or once living organisms of interest. Many species are difficult to study in isolation, because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. DNA sequence-based methods circumvent these obstacles, as DNA can be directly isolated from live or dead cells in a variety of contexts, and have led to the emergence of a new field referred to as metagenomics

Análisis de la jerarquía normativa del DL N° 689 y el Régimen Laboral para Venezolanos, regulado en el artículo 12 del DS N° 001-2018-IN

Perú es considerado como el segundo país en el mundo que acoge a mayor número de migrantes venezolanos, 456 mil es la cifra de ciudadanos venezolanos en Perú respecto a inicios de setiembre del 2018, de los cuales 106 mil ya cuentan con Permiso Temporal de Permanencia, según informe emitido por la Superintendencia Nacional de Migraciones. El gobierno no puede ser, ni ha sido indiferente a ello y, frente a los actuales escenarios es que, a inicios del año 2017, el Poder Ejecutivo, a través de las facultades otorgadas, empezó a emitir normas jurídicas dirigidas a la protección de los ciudadanos venezolanos que ingresaban al territorio nacional; seguido de ello, en enero del presente año, se publicó el DS N° 001-2018, por el cual se aprueban los lineamientos para el otorgamiento del Permiso Temporal de Permanencia para las personas de nacionalidad venezolana, además de regular el Régimen Laboral, el cual ha sido materia de la presente investigación. El presente trabajo toma como ejemplo las diversas investigaciones realizadas en tesis de pre y post grado referente a la regulación de la contratación de los trabajadores extranjeros en el Perú. Así como también, se realizó un análisis de lo regulado en el Decreto Legislativo N° 689 y diversos tratados y convenios internacionales al que Perú se encuentra adscrito. Finalmente, se analizó las normas que se han venido creando en favor de los ciudadanos de nacionalidad venezolana, frente al marco normativo ya existente