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

• 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
• topic: PepYMV; Potyvírus; SI-Gal83; TCTP; Tomate; (PepYMV); Potyvirus; Sl-Gal83; TCTP; Tomato; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

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.