Article ; Online: Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans.
2020 Volume 10, Issue 1, Page(s) 2936
Abstract: To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) ... ...
| Abstract | To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) that is compatible with the original Tet-on plasmid pNIM1, which carries a nourseothricin-resistant marker (CaSAT1). By using GFPmut2 and mCherry as reporters, we found that the two complementary Tet-on plasmids act synergistically in C. albicans with doxycycline in a dose-dependent manner and that expression of the fusion proteins, CaCdc11-GFPmut2 and mCherry-CaCdc10, derived from this system, is septum targeted. Furthermore, to allow detection of protein-protein interactions with the reassembly of a split fluorescent protein, we incorporated mCherry into our system. We generated pWTN1-RN and pNIM1-RC, which express the N-terminus (amino acids 1-159) and C-terminus (amino acids 160-237) of mCherry, respectively. To verify BiFC with mCherry, we created the pWTN1-CDC42-RN (or pWTN1-RN-CDC42) and pNIM1-RC-RDI1 plasmids. C. albicans cells containing these plasmids treated with doxycycline co-expressed the N- and C-terminal fragments of mCherry either N-terminally or C-terminally fused with CaCdc42 and CaRdi1, respectively, and the CaCdc42-CaRdi1 interaction reconstituted a functional form of mCherry. The establishment of this Tet-on-based BiFC system in C. albicans should facilitate the exploration of protein-protein interactions under a variety of conditions. |
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| MeSH term(s) | Biological Assay/methods ; Candida albicans/drug effects ; Candida albicans/genetics ; Candida albicans/metabolism ; Doxycycline/pharmacology ; Fluorescence ; Fungal Proteins/metabolism ; Gene Expression Regulation, Fungal/drug effects ; Genetic Markers ; Hygromycin B/pharmacology ; Luminescent Proteins/metabolism ; Protein Binding/drug effects ; Protein Interaction Mapping ; Septins/metabolism ; Tetracycline/pharmacology |
| Chemical Substances | Fungal Proteins ; Genetic Markers ; Luminescent Proteins ; Hygromycin B (3XQ2233B0B) ; Septins (EC 3.6.1.-) ; Tetracycline (F8VB5M810T) ; Doxycycline (N12000U13O) |
| Language | English |
| Publishing date | 2020-02-19 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, Non-U.S. Gov't |
| ZDB-ID | 2615211-3 |
| ISSN | 2045-2322 ; 2045-2322 |
| ISSN (online) | 2045-2322 |
| ISSN | 2045-2322 |
| DOI | 10.1038/s41598-020-59891-7 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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