Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)

Detalhes bibliográficos
Autor(a) principal: Cascardo, Renan de Souza
Data de Publicação: 2011
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/4734
Resumo: The genomes of most plant viruses code for only 4-10 proteins which are required to complete the infection cycle, including the stages of viral genome replication, cell-to-cell movement and systemic spread. For a successful infection, these viral proteins must interact with host factors, modulating metabolic pathways and coordinating a complex network of biochemical and molecular interactions in the pathogen s favor. A subtractive library constructed from susceptible tomato plants infected by the plant potyvirus Pepper yellow mosaic virus (PepYMV) identified several genes which are putatively involved in the initial steps of the viral infection process, including those that code for the Translationally Controlled Tumor Protein (TCTP) and the tomato homologue of the Saccharomyces cerevisae Gal83 (Sl-Gal83), a protein of the SNF1 complex. TCTP is a highly conserved protein in eukaryotes, and is involved in several fundamental cellular processes such as cell growth, cell cycle progression, programmed cell death and protection against different types of stresses. SNF1 plays an important role in transcriptional activation and expression of genes involved in the cellular response to different stresses, such as nutrient limitation, high salinity, heat shock and virus infection. The objectives of this work were to study the roles of TCTP and Sl-Gal83 during PepYMV infection in susceptible hosts. Transgenic tomatoes (cv. 'Moneymaker') silenced for these genes were generated and were mechanically inoculated with PepYMV. ELISA and qRT-PCR were performed to verify the phenotype resulting from Sl-Gal83 and TCTP silencing. The results showed that non-transformed plants were infected, while all ten silenced plants remained symptomless, were ELISA negative and had reduced viral load. The subcellular localization of TCTP in healthy and PepYMVinfected plants was analyzed by confocal microscopy using a TCTP-YFP fusion. In healthy plants the subcellular localization of TCTP is cytoplasmatic, as described for other organisms. Forty-eight hours after PepYMV infection, TCTP is relocated to the nucleus. To determine which PepYMV protein(s) promotes nuclear targeting of TCTP, each viral protein (P1, HC-Pro, P3, PIPO, P3N-PIPO, CI, 6K2, NIa, NIb and CP) was coexpressed individually with pYFP-TCTP. Results showed that TCTP accumulates predominantly in the nucleus when co-infiltrated with CI and NIa, and is distributed equally in the nucleus and cytoplasm when co-infiltrated with P1, PIPO, P3N-PIPO, 6K2 and NIb. In plants co-infiltrated with HC-Pro, P3 and CP, TCTP remained in the cytoplasm. In order to identify and characterize additional viral and host factors involved in the TCTP-mediated network of plant-potyvirus interactions, TCTP was cloned as a TAP (tandem affinity purification) tag fusion.Protein complexes were purified using TAP, and its constituents were analyzed by mass spectrometry. However,it was not possible to identify the proteins. The probable limiting factor for a more efficient detection was the low concentration of protein eluted. Thus, it is necessary to optimize the protocol of extraction/elution to obtain a higher concentration of proteins complexed with nTAPi- TCTP. Together, the results of this work indicate that both TCTP and Sl-Gal83 play critical roles in the tomato-PepYMV interaction, being necessary for the establishment of a systemic infection. Further studies should be conducted to improve our understanding of the nature and mechanisms of the interactions between TCTP, Sl-Gal83 and PepYMV.
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spelling Cascardo, Renan de Souzahttp://lattes.cnpq.br/7577204500987847Zerbini, Poliane Alfenashttp://lattes.cnpq.br/8632328159533071Carvalho, Claudine Márciahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6Zerbini Júnior, Francisco Murilohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783743U5Queiroz, Marisa Vieira dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z52015-03-26T13:42:18Z2011-11-072015-03-26T13:42:18Z2011-02-28CASCARDO, Renan de Souza. Study of the involvement of the genes that encode the proteins Sl-Gal83 and TCTP in the infection of a susceptible host by Pepper yellow mosaic virus (PepYMV). 2011. 76 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2011.http://locus.ufv.br/handle/123456789/4734The genomes of most plant viruses code for only 4-10 proteins which are required to complete the infection cycle, including the stages of viral genome replication, cell-to-cell movement and systemic spread. For a successful infection, these viral proteins must interact with host factors, modulating metabolic pathways and coordinating a complex network of biochemical and molecular interactions in the pathogen s favor. A subtractive library constructed from susceptible tomato plants infected by the plant potyvirus Pepper yellow mosaic virus (PepYMV) identified several genes which are putatively involved in the initial steps of the viral infection process, including those that code for the Translationally Controlled Tumor Protein (TCTP) and the tomato homologue of the Saccharomyces cerevisae Gal83 (Sl-Gal83), a protein of the SNF1 complex. TCTP is a highly conserved protein in eukaryotes, and is involved in several fundamental cellular processes such as cell growth, cell cycle progression, programmed cell death and protection against different types of stresses. SNF1 plays an important role in transcriptional activation and expression of genes involved in the cellular response to different stresses, such as nutrient limitation, high salinity, heat shock and virus infection. The objectives of this work were to study the roles of TCTP and Sl-Gal83 during PepYMV infection in susceptible hosts. Transgenic tomatoes (cv. 'Moneymaker') silenced for these genes were generated and were mechanically inoculated with PepYMV. ELISA and qRT-PCR were performed to verify the phenotype resulting from Sl-Gal83 and TCTP silencing. The results showed that non-transformed plants were infected, while all ten silenced plants remained symptomless, were ELISA negative and had reduced viral load. The subcellular localization of TCTP in healthy and PepYMVinfected plants was analyzed by confocal microscopy using a TCTP-YFP fusion. In healthy plants the subcellular localization of TCTP is cytoplasmatic, as described for other organisms. Forty-eight hours after PepYMV infection, TCTP is relocated to the nucleus. To determine which PepYMV protein(s) promotes nuclear targeting of TCTP, each viral protein (P1, HC-Pro, P3, PIPO, P3N-PIPO, CI, 6K2, NIa, NIb and CP) was coexpressed individually with pYFP-TCTP. Results showed that TCTP accumulates predominantly in the nucleus when co-infiltrated with CI and NIa, and is distributed equally in the nucleus and cytoplasm when co-infiltrated with P1, PIPO, P3N-PIPO, 6K2 and NIb. In plants co-infiltrated with HC-Pro, P3 and CP, TCTP remained in the cytoplasm. In order to identify and characterize additional viral and host factors involved in the TCTP-mediated network of plant-potyvirus interactions, TCTP was cloned as a TAP (tandem affinity purification) tag fusion.Protein complexes were purified using TAP, and its constituents were analyzed by mass spectrometry. However,it was not possible to identify the proteins. The probable limiting factor for a more efficient detection was the low concentration of protein eluted. Thus, it is necessary to optimize the protocol of extraction/elution to obtain a higher concentration of proteins complexed with nTAPi- TCTP. Together, the results of this work indicate that both TCTP and Sl-Gal83 play critical roles in the tomato-PepYMV interaction, being necessary for the establishment of a systemic infection. Further studies should be conducted to improve our understanding of the nature and mechanisms of the interactions between TCTP, Sl-Gal83 and PepYMV.O genoma da maioria dos vírus que infectam plantas codifica apenas 4-10 proteínas, que são necessárias para completar o ciclo de infecção, incluindo as etapas de replicação do genoma viral, movimento célula-a-célula e movimento sistêmico. Para uma infecção bem sucedida, as proteínas virais devem interagir com fatores do hospedeiro, modulando processos metabólicos e coordenando uma rede complexa de interações bioquímicas e moleculares a favor do patógeno. Uma biblioteca subtrativa foi construída a partir de plantas de tomateiro suscetíveis infectadas pelo potyvírus Pepper yellow mosaic virus (PepYMV). Foram identificados vários genes potencialmente envolvidos nas etapas iniciais do processo de infecção viral. Dentre os genes identificados encontram-se os genes que codificam as proteínas Translationally controlled tumor protein (TCTP) e o homólogo de tomate de Saccharomyces cerevisae Gal83 (Sl-Gal83), uma proteína do complexo Sucrose non-fermenting-1 (SNF1). TCTP é uma proteína altamente conservada em eucariotos, e está envolvida em vários processos celulares básicos, como crescimento celular, progressão do ciclo celular, morte celular programada e proteção contra diversos tipos de estresses. SNF1 desempenha um papel importante na ativação da transcrição e expressão de genes envolvidos na resposta celular aos diferentes tipos de estresses, tais como limitação de nutrientes, salinidade elevada, choque térmico e infecção viral. O objetivo deste trabalho foi estudar o papel das proteínas TCTP e Sl-Gal83 durante o processo de infecção pelo PepYMV em hospedeiros suscetíveis. Plantas transgênicas de tomate cv. Moneymaker silenciadas para estes genes foram produzidas e inoculadas mecanicamente com o PepYMV. O estabelecimento da infecção viral foi avaliado por ELISA e qRT-PCR. Plantas não-transformadas foram infectadas, enquanto as plantas silenciadas permaneceram assintomáticas. Nas plantas silenciadas o ELISA apresentou resultado negativo e a carga viral foi reduzida. A localização subcelular de TCTP em plantas sadias e infectadas pelo PepYMV foi analisada por microscopia confocal utilizando-se uma fusão YFP-TCTP. Em plantas sadias a localização subcelular de TCTP é citoplasmática, conforme descrito para outros organismos. Quarenta e oito horas após a infecção pelo PepYMV, TCTP foi redirecionada para o núcleo. Para determinar qual é a proteína viral que direciona a relocalização de TCTP para o núcleo, cada uma das proteínas virais (P1, HC-Pro, P3, PIPO, P3N-PIPO, CI, 6K2, NIa, NIb e CP) foi coinfiltrada individualmente com pYFP-TCTP. Os resultados mostraram que TCTP acumula predominantemente no núcleo quando co-infiltrada com CI e NIa, e está igualmente distribuída no núcleo e citoplasma, quando co-infiltrada com P1, PIPO, P3NPIPO, 6K2 e NIb. Em plantas co-infiltradas com HC-Pro, P3 e CP, TCTP permaneceu no citoplasma. Para identificar possíveis proteínas virais e/ou do hospedeiro que interagem com TCTP foi realizado um ensaio de purificação por afinidade em tandem. A análise por SDS-PAGE de extratos protéicos de plantas expressando nTAPi-TCTP mostrou duas bandas entre 40 e 50 kDa presentes no extrato purificado. Os fragmentos foram analisados por espectrometria de massa, porém não foi possível identificar as proteínas. Provavelmente o limitante para uma detecção mais eficiente foi a baixa concentração de proteínas eluídas. Com isso, torna-se necessário a otimização do protocolo de extração/eluição para a obtenção de uma maior concentração de proteínas complexadas com nTAPi-TCTP. Em conjunto, os resultados obtidos neste trabalho sugerem que tanto TCTP quanto Sl-Gal83 possuem papéis fundamentais na interação PepYMV-tomate, sendo necessárias para o estabelecimento de uma infecção sistêmica. Estudos adicionais devem ser realizados para melhor compreensão da natureza e dos mecanismos das interações entre TCTP, Sl-Gal83 e o PepYMV.Fundação de Amparo a Pesquisa do Estado de Minas Geraisapplication/pdfporUniversidade Federal de ViçosaMestrado em Genética e MelhoramentoUFVBRGenética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; MePepYMVPotyvírusSI-Gal83TCTPTomate(PepYMV)PotyvirusSl-Gal83TCTPTomatoCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOSEstudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)Study of the involvement of the genes that encode the proteins Sl-Gal83 and TCTP in the infection of a susceptible host by Pepper yellow mosaic virus (PepYMV)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf2907131https://locus.ufv.br//bitstream/123456789/4734/1/texto%20completo.pdf7c8c658c01f5e065741077a77ef12f4dMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain170037https://locus.ufv.br//bitstream/123456789/4734/2/texto%20completo.pdf.txt5885369a208ff1f8630234f81d1ddc76MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3601https://locus.ufv.br//bitstream/123456789/4734/3/texto%20completo.pdf.jpg74f45c1442eb74bf6f99269df9d56fd0MD53123456789/47342016-04-10 23:03:13.01oai:locus.ufv.br:123456789/4734Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:03:13LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
dc.title.alternative.eng.fl_str_mv Study of the involvement of the genes that encode the proteins Sl-Gal83 and TCTP in the infection of a susceptible host by Pepper yellow mosaic virus (PepYMV)
title Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
spellingShingle Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
Cascardo, Renan de Souza
PepYMV
Potyvírus
SI-Gal83
TCTP
Tomate
(PepYMV)
Potyvirus
Sl-Gal83
TCTP
Tomato
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
title_short Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
title_full Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
title_fullStr Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
title_full_unstemmed Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
title_sort Estudo do envolvimento das proteínas Sl-Gal83 e TCTP na infecção de hospedeiros suscetíveis pelo potyvírus Pepper yellow mosaic virus (PepYMV)
author Cascardo, Renan de Souza
author_facet Cascardo, Renan de Souza
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/7577204500987847
dc.contributor.author.fl_str_mv Cascardo, Renan de Souza
dc.contributor.advisor-co1.fl_str_mv Zerbini, Poliane Alfenas
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/8632328159533071
dc.contributor.advisor-co2.fl_str_mv Carvalho, Claudine Márcia
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6
dc.contributor.advisor1.fl_str_mv Zerbini Júnior, Francisco Murilo
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783743U5
dc.contributor.referee1.fl_str_mv Queiroz, Marisa Vieira de
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z5
contributor_str_mv Zerbini, Poliane Alfenas
Carvalho, Claudine Márcia
Zerbini Júnior, Francisco Murilo
Queiroz, Marisa Vieira de
dc.subject.por.fl_str_mv PepYMV
Potyvírus
SI-Gal83
TCTP
Tomate
topic PepYMV
Potyvírus
SI-Gal83
TCTP
Tomate
(PepYMV)
Potyvirus
Sl-Gal83
TCTP
Tomato
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
dc.subject.eng.fl_str_mv (PepYMV)
Potyvirus
Sl-Gal83
TCTP
Tomato
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
description The genomes of most plant viruses code for only 4-10 proteins which are required to complete the infection cycle, including the stages of viral genome replication, cell-to-cell movement and systemic spread. For a successful infection, these viral proteins must interact with host factors, modulating metabolic pathways and coordinating a complex network of biochemical and molecular interactions in the pathogen s favor. A subtractive library constructed from susceptible tomato plants infected by the plant potyvirus Pepper yellow mosaic virus (PepYMV) identified several genes which are putatively involved in the initial steps of the viral infection process, including those that code for the Translationally Controlled Tumor Protein (TCTP) and the tomato homologue of the Saccharomyces cerevisae Gal83 (Sl-Gal83), a protein of the SNF1 complex. TCTP is a highly conserved protein in eukaryotes, and is involved in several fundamental cellular processes such as cell growth, cell cycle progression, programmed cell death and protection against different types of stresses. SNF1 plays an important role in transcriptional activation and expression of genes involved in the cellular response to different stresses, such as nutrient limitation, high salinity, heat shock and virus infection. The objectives of this work were to study the roles of TCTP and Sl-Gal83 during PepYMV infection in susceptible hosts. Transgenic tomatoes (cv. 'Moneymaker') silenced for these genes were generated and were mechanically inoculated with PepYMV. ELISA and qRT-PCR were performed to verify the phenotype resulting from Sl-Gal83 and TCTP silencing. The results showed that non-transformed plants were infected, while all ten silenced plants remained symptomless, were ELISA negative and had reduced viral load. The subcellular localization of TCTP in healthy and PepYMVinfected plants was analyzed by confocal microscopy using a TCTP-YFP fusion. In healthy plants the subcellular localization of TCTP is cytoplasmatic, as described for other organisms. Forty-eight hours after PepYMV infection, TCTP is relocated to the nucleus. To determine which PepYMV protein(s) promotes nuclear targeting of TCTP, each viral protein (P1, HC-Pro, P3, PIPO, P3N-PIPO, CI, 6K2, NIa, NIb and CP) was coexpressed individually with pYFP-TCTP. Results showed that TCTP accumulates predominantly in the nucleus when co-infiltrated with CI and NIa, and is distributed equally in the nucleus and cytoplasm when co-infiltrated with P1, PIPO, P3N-PIPO, 6K2 and NIb. In plants co-infiltrated with HC-Pro, P3 and CP, TCTP remained in the cytoplasm. In order to identify and characterize additional viral and host factors involved in the TCTP-mediated network of plant-potyvirus interactions, TCTP was cloned as a TAP (tandem affinity purification) tag fusion.Protein complexes were purified using TAP, and its constituents were analyzed by mass spectrometry. However,it was not possible to identify the proteins. The probable limiting factor for a more efficient detection was the low concentration of protein eluted. Thus, it is necessary to optimize the protocol of extraction/elution to obtain a higher concentration of proteins complexed with nTAPi- TCTP. Together, the results of this work indicate that both TCTP and Sl-Gal83 play critical roles in the tomato-PepYMV interaction, being necessary for the establishment of a systemic infection. Further studies should be conducted to improve our understanding of the nature and mechanisms of the interactions between TCTP, Sl-Gal83 and PepYMV.
publishDate 2011
dc.date.available.fl_str_mv 2011-11-07
2015-03-26T13:42:18Z
dc.date.issued.fl_str_mv 2011-02-28
dc.date.accessioned.fl_str_mv 2015-03-26T13:42:18Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.citation.fl_str_mv CASCARDO, Renan de Souza. Study of the involvement of the genes that encode the proteins Sl-Gal83 and TCTP in the infection of a susceptible host by Pepper yellow mosaic virus (PepYMV). 2011. 76 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2011.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/4734
identifier_str_mv CASCARDO, Renan de Souza. Study of the involvement of the genes that encode the proteins Sl-Gal83 and TCTP in the infection of a susceptible host by Pepper yellow mosaic virus (PepYMV). 2011. 76 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2011.
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dc.publisher.department.fl_str_mv Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
publisher.none.fl_str_mv Universidade Federal de Viçosa
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