Details
Original language | English |
---|---|
Pages (from-to) | 158-165 |
Number of pages | 8 |
Journal | Journal of virological methods |
Volume | 174 |
Issue number | 1-2 |
Early online date | 5 Apr 2011 |
Publication status | Published - Jun 2011 |
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 (∼3.5. 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.
Keywords
- Bimolecular fluorescence complementation (BiFC), Capsicum chlorosis virus, Plum pox virus, Protein-protein interaction, Red fluorescent protein
ASJC Scopus subject areas
- Immunology and Microbiology(all)
- Virology
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In: Journal of virological methods, Vol. 174, No. 1-2, 06.2011, p. 158-165.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - An optimized mRFP-based bimolecular fluorescence complementation system for the detection of protein-protein interactions in planta
AU - Zilian, Eva
AU - Maiss, Edgar
PY - 2011/6
Y1 - 2011/6
N2 - 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 (∼3.5. 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.
AB - 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 (∼3.5. 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.
KW - Bimolecular fluorescence complementation (BiFC)
KW - Capsicum chlorosis virus
KW - Plum pox virus
KW - Protein-protein interaction
KW - Red fluorescent protein
UR - http://www.scopus.com/inward/record.url?scp=79955769528&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2011.03.032
DO - 10.1016/j.jviromet.2011.03.032
M3 - Article
C2 - 21473882
AN - SCOPUS:79955769528
VL - 174
SP - 158
EP - 165
JO - Journal of virological methods
JF - Journal of virological methods
SN - 0166-0934
IS - 1-2
ER -