RESUMEN
Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, "genome activation" and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members.
Asunto(s)
Ilarvirus/metabolismo , Ilarvirus/patogenicidad , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.
Asunto(s)
Virus del Mosaico , Enfermedades de las Plantas/virología , Solanum lycopersicum/ultraestructura , Solanum lycopersicum/virología , Proteínas de la Cápside/ultraestructura , Chile , Inmunohistoquímica , Solanum lycopersicum/citología , Virus del Mosaico/química , Virus del Mosaico/patogenicidad , Virus del Mosaico/ultraestructura , Hojas de la Planta/ultraestructura , Hojas de la Planta/virología , Potexvirus/química , Potexvirus/ultraestructura , VirulenciaRESUMEN
MAIN CONCLUSION: NO accelerates transition of germinated embryos from heterotrophy to autotrophy by stimulation of chloroplasts maturation. NO-mediated autotrophy of apple seedlings correlates to increased content of RuBisCO small subunit and improvement of parameters of chlorophyll a fluorescence. Nitric oxide (NO) acts as signaling molecule involved in regulation of various physiological processes in plants, although its involvement in cotyledons greening is poorly recognized. To identify the importance of NO signal for plant growth and development we investigated the effects of short-term application of NO at various developmental stages of seedlings of apple (Malus domestica Borkh.) on cotyledons' chlorophyll a to b ratio, chlorophyll a fluorescence, photosynthetic activity, carbohydrates and RuBisCO both subunits content. NO-dependent biochemical alterations were linked to cytological observation of developing plastids in cotyledons of apple plants. Abnormal plantlets developing from dormant apple embryos are characterized by anatomical malformations of cotyledons. Short-term pre-treatment with NO of isolated embryos or seedlings with developmental anomalies resulted in formation of plants with cotyledons of equal size and chlorophyll content; these responses were blocked by NO scavenger. NO independently of time point of application accelerated embryos transition from heterotrophy to autotrophy by stimulation of photosynthetic activity, improvement of parameters of chlorophyll a fluorescence (F v/F m, F v/F 0) and increased content of RuBisCO small subunit. Further analysis showed that NO application modified glucose and hydrogen peroxide concentration in cotyledons. Beneficial effect of NO on development of seedlings without any abnormalities was manifested at ultrastructural level by decline in amount of proplastids and induction of formation and maturation of chloroplasts. Our data suggest that progress of autotrophy of young seedlings is governed by NO acting as stimulator of chloroplast-to-nucleus signaling.
Asunto(s)
Cotiledón/metabolismo , Malus/metabolismo , Óxido Nítrico/metabolismo , Cotiledón/efectos de los fármacos , Malus/efectos de los fármacos , Óxido Nítrico/farmacología , Fotosíntesis/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismoRESUMEN
Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15-30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG-Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.
Asunto(s)
Retículo Endoplásmico/ultraestructura , Orgánulos/ultraestructura , Orgánulos/virología , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Virus ARN/fisiología , Proteínas de la Cápside/aislamiento & purificación , Núcleo Celular/ultraestructura , Núcleo Celular/virología , Cloroplastos/ultraestructura , Cloroplastos/virología , Retículo Endoplásmico/virología , Células del Mesófilo/ultraestructura , Células del Mesófilo/virología , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Mitocondrias/virología , Floema/ultraestructura , Floema/virología , Hojas de la Planta/virología , Virus de Plantas/ultraestructura , Virus ARN/ultraestructura , Solanum tuberosum/virología , Nicotiana/virologíaRESUMEN
The ultrastructural analysis of tobacco, potato and pepper tissues during infection with necrotic strains and the ordinary Potato virus Y strain of revealed the presence of virus inclusions not only in the epidermis and mesophyll but also in the vascular tissues. For the first time cytoplasmic inclusions were documented in companion cells and phloem parenchyma as well as in xylem tracheary elements. The ultrastructural features studied in this work consisted of mostly laminated inclusions (in the traverse and longitudinal section), which were frequently connected with enlarged cisternae of endoplasmic reticulum (ER) located in the direct vicinity of the cell wall attached to virus particles opposite to plasmodesmata. It was noticed that ER participates in synthesis and condensation of the PVY inclusions. During compatible interaction of tobacco and potato plants with PVY, amorphous and nuclear inclusions were observed. Such forms were not found in pepper tissues and potato revealing the hypersensitivity reaction to the infection with PVY necrotic strains. It was stated that the forms of cytoplasmic inclusions cannot serve as a cytological criterion to distinguish the potato virus Y strains and do not depend on host resistance level. Only in compatible interaction in Solanaceous plants tissues cytoplasmic inclusions were observed from the moment the morphological symptoms appeared. In the reaction of hypersensitivity, the inclusions were found on the 24th day following the infection with the PVY necrotic strains, whereas the symptoms were observed 3 days after the PVY infection.
Asunto(s)
Capsicum/ultraestructura , Nicotiana/ultraestructura , Enfermedades de las Plantas/virología , Potyvirus/crecimiento & desarrollo , Solanum tuberosum/ultraestructura , Capsicum/virología , Pared Celular/ultraestructura , Citoplasma/ultraestructura , Cuerpos de Inclusión Viral/ultraestructura , Orgánulos/ultraestructura , Solanum tuberosum/virología , Nicotiana/virologíaRESUMEN
Ca(2+) is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Accumulating evidence suggests that Ca(2+) serves as a messenger in many growth and developmental processes and in plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of cytoplasmic [Ca(2+)](cyt) in plants. The calcium free ions were detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) from 10h to 5 days after a Potato virus Y (PVY) infection. Calcium was detected in vivo by its reaction with Alizarin S Red, producing an intense red staining in contact with calcium free ions. Calcium was found in the necrotic area of the epidermal and mesophyll cells 3 days after the PVY infection (when morphological symptoms on potato leaves appeared). Calcium ions were detected cytochemically in HR also by its reaction with potassium pyroantimonate. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised increased accumulation of electron-dense deposits of calcium pyroantimonate were detected. Calcium deposition was observed in necrotic and non-necrotic areas, starting from 10h after PVY infection. The deposits were present along ER cisternae, chloroplasts and mitochondria envelopes connected with PVY particles. The precipitates of calcium antimonate were detected near the nuclear envelope, inside karyolymph and along tracheary elements, especially when virus particles were present inside.
Asunto(s)
Calcio/análisis , Cationes Bivalentes/análisis , Potyvirus/inmunología , Solanum tuberosum/química , Solanum tuberosum/inmunologíaRESUMEN
The reactive oxygen species hydrogen peroxide (H(2)O(2)) was detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) 24 and 48 h after a Potato virus Y (PVY) infection. Hydrogen peroxide was detected in vivo by its reaction with 3.3-diaminobenzidine, producing a reddish-brown staining in contact with H(2)O(2). Hydrogen peroxide was detected in the necrotic area of the epidermal and mesophyll cells 24 and 48 h after the PVY infection. Highly localised accumulations of H(2)O(2) were found within xylem tracheary elements, and this was much more intensive than in non-infected leaves. Hydrogen peroxide was detected cytochemically in HR also by its reaction with cerium chloride, producing electron-dense deposits of cerium perhydroxides. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised accumulations of H(2)O(2) was detected in plant cell walls. The most intensive accumulation was present in the bordering cell walls of necrotic mesophyll cells and the adjacent non-necrotic mesophyll cells. Intensive electron-dense deposits of cerium perhydroxide were found along ER cistrenae and chloroplast envelopes connected with PVY particles. The precipitates of hydrogen peroxide were detected in the nuclear envelope and along tracheary elements, especially when virus particles were present inside. The intensive accumulation of H(2)O(2) at the early stages of potato-PVY interaction is consistent with its role as an antimicrobial agent and for this reason it has been regarded as a signalling molecule.
Asunto(s)
Peróxido de Hidrógeno/análisis , Potyvirus/crecimiento & desarrollo , Solanum tuberosum/química , Solanum tuberosum/virología , Solanum tuberosum/inmunología , Factores de TiempoRESUMEN
Annexin A6 (AnxA6), calcium- and membrane-binding protein, is involved in membrane dynamics. It exists in the cell in two isoforms, AnxA6-1 and AnxA6-2, varying only by the VAAEIL sequence. In most cells, AnxA6-1 predominates. A limited number of observations suggests that both isoforms differ from each other functionally. The EGF-dependent Ca(2+) influx in A431 cells is inhibited only by AnxA6-1. Moreover, AnxA6-2 was found to exhibit higher affinity for Ca(2+). In this report we addressed the potential significance of the VAAEIL deletion in AnxA6-2. For this purpose, we expressed AnxA6 isoform cDNAs in bacteria or mouse Balb/3T3 fibroblasts. The recombinant AnxA6-2 was characterized by a less extended molecular shape than that of AnxA6-1 and required a narrower [Ca(2+)] range to bind liposomes. Upon lowering pH in the presence of EGTA recombinant AnxA6-2 became less hydrophobic than AnxA6-1 as revealed by the Triton X-114 partition. Furthermore, AnxA6-2 revealed stronger F-actin binding than that of AnxA6-1. Immunofluorescence microscopy showed that the EGFP-tagged AnxA6 isoforms expressed in Balb/3T3 fibroblasts relocate in a Ca(2+)- and H(+)-sensitive manner to the vesicular structures in a perinuclear region or in cytosol. Cell fractionation showed that in resting conditions AnxA6-1 is associated with early endosomes and AnxA6-2 with late endosomes, and an increase in [Ca(2+)] and/or [H(+)] induced their opposite distribution. These findings suggest a potentially independent regulation, localization, and function of AnxA6 isoforms in Balb/3T3 fibroblasts. More generally, our findings suggest distinct functions of AnxA6 isoforms in membrane dynamics.