Potyvirus: caracterização parcial de espécies em plantas daninhas associadas a cultura do pimentão, avaliação de genótipos de alface e análise subcelular do eIF4E e de proteínas do Lettuce mosaic virus

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Caracterização e ocorrência de bidens mosaic virus (bimv) em regiões produtoras de alface no Estado de São Paulo

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Biological and molecular characterisation of Bidens mosaic virus supports its assignment as a member of a distinct species in the genus Potyvirus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudio etiopatogénico del circovirus porcino tipo 2 asociado a virus y bacterias en una infección natural

Programa de doctorado: Sanidad y patología animal

Fate of hairpin transcript components during RNA silencing and its suppression in transgenic virus-resistant tobacco

Transgenic tobacco plants, carrying a Potato virus Y (PVY)-NIa hairpin sequence separated by a unique unrelated spacer sequence were specifically silenced and highly resistant to PVY infection. In such plants neither PVY-NIa nor spacer transgene transcripts were detectable by specific quantitative real time reverse transcriptase PCR (RT-qPCR) assays of similar relative efficiencies developed for direct comparative analysis. However, small interfering RNAs (siRNAs) specific for the PVY sequence of the transgene and none specific for the LNYV spacer sequence were detected. Following infection with Cucumber mosaic virus (CMV), which suppresses dsRNA-induced RNA silencing, transcript levels of PVY-NIa as well as spacer sequence increased manifold with the same time course. The cellular abundance of the single-stranded (ss) spacer sequence was consistently higher than that of PVY dsRNA in all cases. The results show that during RNA silencing and its suppression of a hairpin transcript in transgenic tobacco, the ssRNA spacer sequence is affected differently than the dsRNA. In PVY-silenced plants. the spacer is efficiently degraded by a mechanism not involving the accumulation of siRNAs, while following suppression of RNA silencing by CMV, the spacer appears protected from degradation. Crown Copyright (c) 2006 Published by Elsevier B.V. All rights reserved.

Análise comparativa da região codificadora para a proteína capsidial de isolados de PepYMV e PVY coletados em pimentão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variabilidade de potyvirus infectando Capsicum spp. no estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise molecular da proteína HC-Pro (Helper Component-Proteinase) e seu papel na relação patógeno-hospedeiro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I β-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

Three-dimensional model of the potyviral genome-linked protein.

The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm – with or without anchoring them in the plasma membrane –, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised neither by adding a C-terminal stabilisation signal nor by anchoring the protein at the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Sweet potato chlorotic stunt virus: Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

Virus : qué son y cómo actúan.

No consta el número de depósito legal

Efecto de la infección por virus persistentes y no persistentes sobre el comportamiento alimenticio, la preferencia, tasa de aterrizaje y asentamiento y la eficacia biológica de Aphis gossypii Glover

En el complejo de plagas que atacan a los principales cultivos hortícolas protegidos, destacan principalmente los Hemípteros, y dentro de estos los pulgones, dada su importancia como vectores de virus que provocan considerables daños y pérdidas económicas. Debido a que la dispersión de la mayoría de los virus de plantas puede ser eficaz con densidades bajas de vectores y su control es muy complicado al no existir métodos curativos para su control, es necesario generar nuevos conocimientos sobre las interacciones virus-vector con el fin de desarrollar nuevas y eficaces estrategias de control. Por ello, el objetivo general de esta Tesis ha sido conocer el efecto de la infección viral (directo-mediado por la presencia del virus en el vector- e indirecto-mediado por las alteraciones físico-químicas que se originan en la planta como consecuencia de la infección viral-) sobre el comportamiento y eficacia biológica del vector Aphis gossypii Glover y sus posibles repercusiones en la epidemiología de virosis de transmisión no persistente (Cucumber mosaic virus, CMV, Cucumovirus) y persistente (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). El primer objetivo de esta Tesis Doctoral, se centró en el estudio del efecto indirecto del virus de transmisión no persistente CMV sobre el comportamiento alimenticio y la preferencia del pulgón A. gossypii en el cultivo de pepino. Los ensayos de despegue y aterrizaje mostraron que los pulgones que fueron liberados en las plantas de pepino infectadas con CMV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (60, 120 y 180 minutos después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CMV). El estudio de preferencia y asentamiento mostró que el vector A. gossypii prefiere asentarse en plantas infectadas con CMV en una etapa temprana de evaluación (30 minutos después de la liberación). Sin embargo, este comportamiento se revirtió en una etapa posterior (4 y 48 horas después de la liberación), donde los pulgones se asentaron más en las plantas no infectadas. A través de la técnica de Gráficos de Penetración Eléctrica (EPG) se observó un efecto indirecto del virus CMV, revelado por un cambio brusco en el comportamiento de prueba del pulgón a lo largo del tiempo, cuando éstos fueron expuestos a las plantas infectadas con CMV. Los primeros 15 minutos de registro EPG mostraron que los pulgones hicieron un número mayor de punciones intracelulares (potencial drops - pds) y pruebas en las plantas infectadas con CMV que en las plantas no infectadas. Por otra parte, la duración de la primera prueba fue más corta y la duración total de las pds por insecto fue mucho más larga en las plantas infectadas con CMV. Se observaron diferencias significativas en el tiempo transcurrido desde el final de la última pd hasta el final de la prueba, siendo ese tiempo más corto para los pulgones que estaban alimentándose en plantas infectadas con CMV. En la segunda hora de registro los pulgones rechazaron las plantas infectadas con CMV como fuente de alimento, permaneciendo menos tiempo en las fases de prueba en floema (fase de salivación – E1 y fase de ingestión del floema – E2). El comportamiento alimenticio observado sobre las plantas infectadas con CMV favorece la adquisición y posterior transmisión de los virus de transmisión no persistente, los cuales son adquiridos e inoculados durante la realización de pruebas intracelulares en las primeras pruebas de corta duración. En el segundo objetivo de la Tesis se evaluó el efecto directo e indirecto del virus de transmisión persistente CABYV en el comportamiento alimenticio y preferencia del pulgón A. gossypii en cultivo de pepino, especie susceptible al virus, y algodón, especie inmune al virus. No se observó un efecto directo del virus relevante en el comportamiento alimenticio del vector, ya que los resultados obtenidos a nivel floemático en plantas de pepino no se observaron en plantas de algodón, inmune al virus CABYV. Esto sugiere que los resultados obtenidos en pepino, pueden deberse a un “posible efecto indirecto” originado por la infección de las plantas susceptibles al virus durante la realización del ensayo, lo que indirectamente puede modificar el comportamiento del pulgón durante la fase de evaluación. Sin embargo, el virus CABYV modificó indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada. Los pulgones tardaron menos tiempo en llegar al floema, realizaron un mayor número de pruebas floemáticas y permanecieron durante más tiempo en actividades floemáticas en plantas infectadas con CABYV. El comportamiento observado sobre las plantas infectadas con CABYV favorece la adquisición de virus persistentes, los cuales son adquiridos durante la alimentación sostenida en floema. El estudio de preferencia y asentamiento de A. gossypii mostró que los pulgones virulíferos prefieren asentarse en plantas no infectadas a corto y largo plazo de evaluación (2, 4 y 48 horas después de la liberación). Los ensayos de despegue y aterrizaje mostraron que los pulgones virulíferos que fueron liberados en las plantas de pepino infectadas con CABYV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (3, 6, 24 y 48 horas después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CABYV). Sin embargo, los pulgones no virulíferos no mostraron preferencia por plantas de pepino no infectadas o infectadas con CABYV en ninguno de los ensayos (preferencia o despegue) o periodos evaluados (corto y largo plazo). Los resultados indican que el virus CABYV es capaz de modificar indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada, favoreciendo su adquisición por su principal vector, A. gossypii. Una vez que los pulgones tienen capacidad de transmitir el virus (virulíferos) se produce un cambio en su comportamiento prefiriendo asentarse sobre plantas no infectadas optimizándose así la dispersión viral. El tercer objetivo de la Tesis, fue evaluar los efectos directos e indirectos del virus CABYV así como los efectos indirectos del virus CMV en la eficacia biológica del vector A. gossypii. Los resultados obtenidos en los ensayos realizados con el virus persistente CABYV indican que el virus parece no modificar directamente ni indirectamente la eficacia biológica del vector en plantas de pepino o algodón, no observándose diferencias estadísticas en ninguno de los parámetros poblacionales evaluados (tiempo de desarrollo, tasa intrínseca de crecimiento, tiempo generacional medio, tasa media de crecimiento relativo y ninfas totales). En cuanto a los ensayos realizados con el virus no persistente, CMV, los resultados muestran un efecto indirecto del virus sobre la biología del vector. Así resultó que tanto la tasa intrínseca de crecimiento natural (rm) como la tasa media de crecimiento relativo (RGR) fueron más altas para pulgones crecidos sobre plantas infectadas con CMV que sobre plantas no infectadas, favoreciendo la reproducción y crecimiento poblacional del vector sobre plantas infectadas con CMV. Los resultados obtenidos en la presente Tesis, ofrecen un ejemplo de como los virus de plantas pueden manipular directa e indirectamente a su vector, maximizando así su dispersión entre las plantas. Esos nuevos conocimientos generados tienen implicaciones importantes en la transmisión, dispersión y en la epidemiología de los virus y deben ser considerados para diseñar o ajustar los modelos de simulación existentes y patrones de dispersión que describen las epidemias de estos virus. ABSTRACT The main objective of this Thesis has been to understand the effect of the viral infection (direct-mediated by the presence of the virus in the vector and indirect mediated by the chemical and physical changes originated in the plant as a consequence of the viral infection) on the behaviour and biological efficacy of the vector Aphis gossypii Glover and its consequences in the epidemiology of two viral diseases, one with non-persistent transmission (Cucumber mosaic virus, CMV, Cucumovirus) and another with persistent transmission (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). The first objective of this Thesis was the study of the indirect effect of the nonpersistent virus CMV on the feeding behaviour and preference of the aphid A. gossypii in cucumber plants. The results of the alighting and settling behaviour studies showed that aphids exhibited no preference to migrate from CMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), but showed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min after release. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected over mockinoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage and aphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph (EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of short superficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (second hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much less time spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour including an early increase in the number of short superficial probes and intracellular punctures followed by a phloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in a NP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting, settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spread of the virus. The second objective of this work was to evaluate the effects that the persistently aphid transmitted Cucurbit aphid-borne yellows virus (CABYV) can induce directly and indirectly on the alighting, settling and probing behaviour activities of the cotton aphid A. gossypii. Only minor direct changes on aphid feeding behaviour was observed due to CABYV when viruliferous aphids fed on mock-inoculated plants. However, the feeding behaviour of non-viruliferous aphids was very different on CABYV-infected than on mockinoculated plants. Non-viruliferous aphids spent longer time feeding from the phloem when plants were infected by CABYV than on mock-inoculated plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. The vector alighting and settling preference was compared between nonviruliferous and viruliferous aphids. Viruliferous aphids showed a clear preference for mockinoculated over CABYV-infected plants at short and long time, while such behaviour was not observed for non-viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimize the transmission and spread of the virus. The third objective was to evaluate the direct and indirect effects of CABYV and indirect effects of the CMV on the A. gossypii fitness. Obtained results for the persistent virus CABYV showed that the virus did not modify the vector fitness in cucumber or cotton plants. None of the evaluated variables was statistically significant (development time (d), intrinsic growth rate (rm), mean relative growth rate (RGR) and total number of nymphs). On the other hand, data obtained for the non-persistent virus (CMV) showed an indirect effect of the virus on the vector fitness. Thus, the rm and RGR were higher for aphids grown on CMV-infected plants compared to aphids grown on mock-inoculated plants. Overall, the obtained results are clear examples of how plant viruses could manipulate directly and indirectly vector behaviour to optimize its own dispersion. These results are important for a better understanding of transmission, dispersion and epidemiology of plant viruses transmitted by vectors. This information could be also considered to design or adjust simulation models and dispersion patterns that describe plant virus epidemics.