Caracterização funcional de uma provável nucleoporina que interage com a proteína NSP de geminivírus

• Autor(a) principal: Fontenelle, Mariana Rodrigues
• Data de Publicação: 2006
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2399
• topic: Nucleoporina; Proteína NSP; Geminivírus; Nucleoporin; NSP protein; Geminivirus; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

The family Geminiviridae is a diverse family of plant virus. The Cabbage leaf curl virus (CaLCuV) of Begomovirus genus possess two genomic components of singlestranded DNA, DNA-A and DNA-B. The DNA-A is required for replication, transactivaction and encapsidation of the viral genome. The DNA-B is required for the virus movement, in which NSP is coded. NSP transports the viral DNA from the nucleus to the cytoplasm and has been demonstrated to interact with a plasma membrane receptor-like kinase, designated NIK (NSP-interacting kinase). In this study, we performed a two-hybrid screening for CaLCuV NSP-interacting proteins, using a cDNA library from Arabidopsis and we isolated a nucleoporin-like protein, designated NAP (NSP-Associated Protein). Further more, it was proved in vitro assays that these proteins interact with each other. The full-length protein, which is encoded by the At4g13350 gene, possess two NAP homologues of unknown functions, At1g08680 and At4g32630 (33% and 31% sequence identity, respectively), in the Arabidopsis genome. Other homologue is human Rip protein that interacts with the HIV Rev protein. To assess directly the significance of NAP-NSP interaction, we identified a mutant in the NAP gene and selected for homozygous Arabidopsis mutant line by PCR. The inactivation of this gene was confirmed by RT-PCR, confirming it is null allele. Wild type Col-0 plants and nap mutant lines were inoculated with CaLCuV DNA-A and DNA-B. Both Col-0 and nap lines developed typical symptoms of CaLCuV infection at similar intensity. Inactivation of the NAP gene does not seem to affect CaLCuV infection in Arabidopsis. These results indicate that either the NAP-NSP interaction is not required for NSP function or the NAP homologs are functionally analogs that are capable to replace the NAP role in the null allele mutant. Over expressing NAP plants were also inoculated with the virus CaLCuV, and presented a discrete increase in plant susceptibility, showing more severe symptoms and higher percentage of infected plants compared with wild type.