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1.
Plant J ; 103(6): 2263-2278, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32593210

RESUMEN

Potato tuber formation is a secondary developmental programme by which cells in the subapical stolon region divide and radially expand to further differentiate into starch-accumulating parenchyma. Although some details of the molecular pathway that signals tuberisation are known, important gaps in our knowledge persist. Here, the role of a member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family (termed StCEN) in the negative control of tuberisation is demonstrated for what is thought to be the first time. It is shown that reduced expression of StCEN accelerates tuber formation whereas transgenic lines overexpressing this gene display delayed tuberisation and reduced tuber yield. Protein-protein interaction studies (yeast two-hybrid and bimolecular fluorescence complementation) demonstrate that StCEN binds components of the recently described tuberigen activation complex. Using transient transactivation assays, we show that the StSP6A tuberisation signal is an activation target of the tuberigen activation complex, and that co-expression of StCEN blocks activation of the StSP6A gene by StFD-Like-1. Transcriptomic analysis of transgenic lines misexpressing StCEN identifies early transcriptional events in tuber formation. These results demonstrate that StCEN suppresses tuberisation by directly antagonising the function of StSP6A in stolons, identifying StCEN as a breeding marker to improve tuber initiation and yield through the selection of genotypes with reduced StCEN expression.


Asunto(s)
Proteínas de Plantas/fisiología , Tubérculos de la Planta/crecimiento & desarrollo , Solanum tuberosum/crecimiento & desarrollo , Genes de Plantas , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente , Solanum tuberosum/metabolismo , Transcriptoma
2.
Planta ; 236(4): 1305-13, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22718313

RESUMEN

Bovine papillomavirus type 1 (BPV-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. Given that the BPV-1 L1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in Nicotiana benthamiana as a prelude to producing a candidate vaccine. It was found that plant codon optimization of L1 gave higher levels of expression than its non-optimized counterpart. Following protein extraction, we obtained high yields (183 mg/kg fresh weight leaf tissue) of relatively pure L1, which had self-assembled into virus-like particles (VLPs). We found that these VLPs elicited a highly specific and strong immune response, and therefore they may have utility as a potential vaccine. This is the first report demonstrating the viable production of a candidate BPV vaccine protein in plants.


Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Papillomavirus Bovino 1/inmunología , Proteínas de la Cápside/inmunología , Nicotiana/metabolismo , Vacunas de Partículas Similares a Virus/inmunología , Animales , Antígenos Virales/aislamiento & purificación , Antígenos Virales/metabolismo , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/metabolismo , Bovinos , Expresión Génica , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Conejos , Proteínas Recombinantes , Nicotiana/genética , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Vacunas de Partículas Similares a Virus/metabolismo , Vacunas de Partículas Similares a Virus/ultraestructura
3.
J Exp Bot ; 62(1): 371-81, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20855456

RESUMEN

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.


Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Manipulación de Alimentos , Pectinas/química , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/enzimología , Solanum tuberosum/enzimología , Hidrolasas de Éster Carboxílico/genética , Pectinas/metabolismo , Proteínas de Plantas/genética , Tubérculos de la Planta/química , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Solanum tuberosum/química , Solanum tuberosum/genética , Solanum tuberosum/metabolismo
4.
Methods Mol Biol ; 451: 477-90, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18370275

RESUMEN

The generation of infectious clones is routinely the first step for reverse genetic studies of RNA plant virus gene and sequence function. The procedure given here, details the creation of cDNA clones of tobacco mosaic virus, from which infectious transcripts can be generated in vitro with T7 RNA polymerase. The procedure describes methods for virion purification, viral RNA extraction, reverse transcription, PCR amplification of genomic cDNA fragments, generation of a full-length cDNA clone under the control of a T7 promoter, in vitro transcription, and infectivity testing.


Asunto(s)
Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/patogenicidad , Clonación Molecular/métodos , Cartilla de ADN , ADN Complementario/genética , ARN Polimerasas Dirigidas por ADN/genética , Amplificación de Genes , Enfermedades de las Plantas/virología , Regiones Promotoras Genéticas , Virus ARN/genética , Virus ARN/patogenicidad , ARN Viral/genética , ARN Viral/aislamiento & purificación , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Nicotiana/virología , Transcripción Genética , Proteínas Virales/genética
5.
J Cell Biol ; 201(7): 981-95, 2013 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-23798728

RESUMEN

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5' end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.


Asunto(s)
Proteínas de Movimiento Viral en Plantas/fisiología , Plasmodesmos/virología , Potexvirus/fisiología , Replicación Viral/fisiología , Transporte Biológico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Modelos Biológicos , Mutación , Proteínas de Movimiento Viral en Plantas/análisis , Proteínas de Movimiento Viral en Plantas/genética , ARN Viral/análisis , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/análisis , ARN Polimerasa Dependiente del ARN/metabolismo , Nicotiana/virología
6.
Front Plant Sci ; 3: 290, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23269927

RESUMEN

The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1-2 µm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

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