An optimized mRFP-based bimolecular fluorescence complementation system for the detection of protein-protein interactions in planta.
J Virol Methods
; 174(1-2): 158-65, 2011 Jun.
Article
em En
| MEDLINE
| ID: mdl-21473882
ABSTRACT
An existing bimolecular fluorescence complementation (BiFC) system, based on a monomeric red fluorescent protein (mRFP), has been optimized for the investigation of protein-protein interactions in planta. The expression plasmids, encoding the N-terminal amino acids (aa) 1-168 and the C-terminal aa 169-225 of the mRFP, allow N- or C-terminal fusion of a split mRFP, with the genes of interest. Two major improvements over the original vectors have been made. Firstly, the coding sequence of a GGGSGGG-linker has been integrated between mRFP sequences and the genes of interest. Secondly, a modified mini binary vector (â¼3.5 kb) was introduced as the backbone for the plant expression plasmids. Based on the results of yeast two-hybrid studies with plant viral proteins, interaction of viral proteins was tested in Nicotiana benthamiana plants and monitored by confocal laser scanning microscopy (CLSM). Plum pox virus coat protein and mutants thereof served as controls. The system was validated using the N-protein of Capsicum chlorosis virus for which a self-interaction was shown for the first time, the Tobacco mosaic virus coat protein and BC1 and BV1 of the Tomato yellow leaf curl Thailand virus. This optimized BiFC system provides a convenient alternative to other BiFC, as well as yeast two-hybrid assays, for detecting protein-protein interactions.
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Base de dados:
MEDLINE
Assunto principal:
Nicotiana
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Mapeamento de Interação de Proteínas
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Proteínas do Capsídeo
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article