Publication: Interacción de la TGB1 del virus del mosaico del pepino dulce (PepMV) con factores de transcripción de tomate
Authors
Rodríguez Úbeda, Jesús Enmanuel
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Escuela Internacional de Doctorado
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Aranda Regules, Miguel Ángel
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
En esta tesis doctoral se ha estudiado la proteína 1 del bloque de tres genes (TGB) del virus del mosaico del pepino dulce (PepMV; especie Potexvirus pepini) con un interés especial en su papel en el núcleo. PepMV, perteneciente al género Potexvirus de la familia Alphaflexiviridae, es actualmente el agente causante de una pandemia en tomate cultivado, con importantes pérdidas económicas asociadas. Además de su relevancia agronómica, PepMV se ha consolidado como un modelo de referencia en virología vegetal gracias al desarrollo de numerosas herramientas biotecnológicas y la caracterización de su genoma e interacción con el huésped. La TGB1 de PepMV desempeña un papel central en distintas etapas del ciclo infectivo, incluyendo el silenciamiento de ARN, aunque muchas de sus funciones han sido inferidas por homología con virus relacionados.
El objetivo de esta tesis doctoral ha sido caracterizar experimentalmente la localización subcelular de la TGB1 de PepMV, con especial interés en su función nuclear, identificando y caracterizando factores nucleares del huésped interactores de TGB1.
En la primera parte de esta tesis se caracterizó la localización subcelular de la TGB1 de PepMV, determinando que se trata de una proteína soluble con una distribución núcleo-citoplásmica en células sanas, con exclusión del nucleolo, que durante la infección se acumula de forma preferente en plasmodesmos y orgánulos de replicación viral (VROs). Aunque en el VRO se concentran gran parte de las funciones asociadas la infección, la presencia consistente de TGB1 en el núcleo sugiere un papel funcional adicional en este compartimento subcelular.
Para abordar esta cuestión, en la segunda parte de esta tesis doctoral, se caracterizaron factores nucleares del huésped interactores de la TGB1 de PepMV identificados a través de un escrutinio de doble híbrido en levadura. El análisis in silico de las secuencias obtenidas permitió identificar las proteínas SlALOG1 y SlbZIP42, pertenecientes a las familias de factores de transcripción ALOG y bZIP, respectivamente. Mientras que la familia bZIP participa en una amplia variedad de procesos biológicos, incluyendo respuesta a estrés tanto abiótico como biótico, la familia ALOG únicamente ha sido descrita como reguladores homeóticos del desarrollo.
Tanto SlALOG1 como SlbZIP42 mostraron localización nuclear en células sanas, donde colocalizaron con la TGB1 de PepMV, y se acumularon de forma preferente en los VROs durante la infección. El análisis comparativo con otros miembros de ambas familias mostró un comportamiento diferencial. Únicamente SlALOG1, dentro de la familia ALOG, fue capaz de acumularse en los VROs, mientras que varios miembros del clado I de la familia bZIP, incluyendo SlbZIP42, pero no SlbZIP11, presentaron esta capacidad, lo que sugiere funciones específicas y no redundantes durante la infección por PepMV. Los ensayos de susceptibilidad a PepMV mostraron una acumulación viral significativamente menor en plantas slalog1 y slbzip42 respecto a plantas de genotipo WT. Además, los análisis transcriptómicos en mutantes slalog1 y slbzip42 mostraron un papel para ambas proteínas como reguladores maestros de la transcripción, implicados principalmente en procesos redox y de regulación hormonal.
In this doctoral thesis, the triple gene block protein 1 (TGB1) of pepino mosaic virus (PepMV; species Potexvirus pepini) was investigated, with particular emphasis on its role in the nucleus. PepMV, a member of the genus Potexvirus within the family Alphaflexiviridae, is currently responsible for a pandemic in cultivated tomato, causing substantial economic losses. Beyond its agronomic relevance, PepMV has become a reference model in plant virology owing to the development of a complete biotechnological toolkit and the characterization of its genome and host interactions. PepMV TGB1 plays a central role in multiple stages of the viral infection cycle, including RNA silencing suppression; however, many of its functions have largely been inferred from homology with related viruses. The main objective of this doctoral thesis was to experimentally characterize the subcellular localization of PepMV TGB1, with particular interest in its nuclear function, through the identification and characterization of nuclear host factors interacting with this viral protein. In the firs part of this thesis, the subcellular localization of PepMV TGB1 was characterized, revealing that it is a soluble protein displaying a nucleo-cytoplasmic distribution in healthy cells, with exclusion from the nucleolus, and that it preferentially accumulates in plasmodesmata and viral replication organelles (VROs) during infection. Although VROs concentrate most functions associated with viral infection, the consistent presence of TGB1 in the nucleus suggests an additional functional role in this subcellular compartment. To address this question, the second part of this thesis focused on the characterization of nuclear host factors interacting with PepMV TGB1, identified through a yeast two-hybrid scree. In silico analysis of the retrieved sequences identified the proteins SlALOG1 and SlbZIP42, members of the ALOG and bZIP transcripcion factor families, respectively. Whereas the bZIP family is involved in a wide range of biological processes, including responses to both biotic and abiotic stresses, the ALOG family has so far been described mainly as homeotic regulators of development. Both SlALOG1 and SlbZIP42 exhibited nuclear localization in healthy cells, where they colocalized with PepMV TGB1, and preferentially accumulated in VROs during infection. Comparative analyses with other members of both families revealed differential behaviors. Within the ALOG family, only SlALOG1 was able to accumulate in VROs, whereas several members of the clade I bZIP family, including SlbZIP42 but not SlbZIP11, displayed this ability, suggesting specific and non-redundant functions during PepMV infection. Susceptibility assays showed significantly lower PepMV accumulation in slalog1 and slbzip42 plants compared with wild-type plants. Furthermore, transcriptomic analyses in slalog1 and slbzip42 mutants indicated that both proteins act as transcriptional master regulators, primarily involved in redox-related processes and hormonal regulation. Based on these results, an integrative model is proposed in which PepMV TGB1 modulates the function and localization of SlALOG1 and members of the bZIP family throughout the infection cycle. During the early stages of infection, TGB1 exhibits a nucleo-cytoplasmic localization without detectable alterations in cellular ultrastructure, coexisting in the nucleus with SlALOG1 without interaction, while constitutively associating with SlbZIP42 and oter bZIP factors. As infection progresses, TGB1 accumulates in nascent viral replication complexes (VRCs) together with bZIP factors, while, in parallel, conformational changes in one or more proteins within the nucleus may enable the TGB1-SlALOG1 interaction. The sequestration of SlALOG1 and bZIP factors by TGB1 would contribute to the modulation of host defense responses. At advances stages of infection, TGB1 would concentrate SlALOG1-containing complexes within VRCs -condensed substructures inside VROs- together with other viral proteins to optimize replication/transcription processes, whereas clade I bZIP family members would form the primary scaffold of these viral organelles.
In this doctoral thesis, the triple gene block protein 1 (TGB1) of pepino mosaic virus (PepMV; species Potexvirus pepini) was investigated, with particular emphasis on its role in the nucleus. PepMV, a member of the genus Potexvirus within the family Alphaflexiviridae, is currently responsible for a pandemic in cultivated tomato, causing substantial economic losses. Beyond its agronomic relevance, PepMV has become a reference model in plant virology owing to the development of a complete biotechnological toolkit and the characterization of its genome and host interactions. PepMV TGB1 plays a central role in multiple stages of the viral infection cycle, including RNA silencing suppression; however, many of its functions have largely been inferred from homology with related viruses. The main objective of this doctoral thesis was to experimentally characterize the subcellular localization of PepMV TGB1, with particular interest in its nuclear function, through the identification and characterization of nuclear host factors interacting with this viral protein. In the firs part of this thesis, the subcellular localization of PepMV TGB1 was characterized, revealing that it is a soluble protein displaying a nucleo-cytoplasmic distribution in healthy cells, with exclusion from the nucleolus, and that it preferentially accumulates in plasmodesmata and viral replication organelles (VROs) during infection. Although VROs concentrate most functions associated with viral infection, the consistent presence of TGB1 in the nucleus suggests an additional functional role in this subcellular compartment. To address this question, the second part of this thesis focused on the characterization of nuclear host factors interacting with PepMV TGB1, identified through a yeast two-hybrid scree. In silico analysis of the retrieved sequences identified the proteins SlALOG1 and SlbZIP42, members of the ALOG and bZIP transcripcion factor families, respectively. Whereas the bZIP family is involved in a wide range of biological processes, including responses to both biotic and abiotic stresses, the ALOG family has so far been described mainly as homeotic regulators of development. Both SlALOG1 and SlbZIP42 exhibited nuclear localization in healthy cells, where they colocalized with PepMV TGB1, and preferentially accumulated in VROs during infection. Comparative analyses with other members of both families revealed differential behaviors. Within the ALOG family, only SlALOG1 was able to accumulate in VROs, whereas several members of the clade I bZIP family, including SlbZIP42 but not SlbZIP11, displayed this ability, suggesting specific and non-redundant functions during PepMV infection. Susceptibility assays showed significantly lower PepMV accumulation in slalog1 and slbzip42 plants compared with wild-type plants. Furthermore, transcriptomic analyses in slalog1 and slbzip42 mutants indicated that both proteins act as transcriptional master regulators, primarily involved in redox-related processes and hormonal regulation. Based on these results, an integrative model is proposed in which PepMV TGB1 modulates the function and localization of SlALOG1 and members of the bZIP family throughout the infection cycle. During the early stages of infection, TGB1 exhibits a nucleo-cytoplasmic localization without detectable alterations in cellular ultrastructure, coexisting in the nucleus with SlALOG1 without interaction, while constitutively associating with SlbZIP42 and oter bZIP factors. As infection progresses, TGB1 accumulates in nascent viral replication complexes (VRCs) together with bZIP factors, while, in parallel, conformational changes in one or more proteins within the nucleus may enable the TGB1-SlALOG1 interaction. The sequestration of SlALOG1 and bZIP factors by TGB1 would contribute to the modulation of host defense responses. At advances stages of infection, TGB1 would concentrate SlALOG1-containing complexes within VRCs -condensed substructures inside VROs- together with other viral proteins to optimize replication/transcription processes, whereas clade I bZIP family members would form the primary scaffold of these viral organelles.
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