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

Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC

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