Gateway-compatible inducible vector set for the functional analysis of transcription factors in plants
The inducible vectors pER8–Gateway–3Flag and pER8–Gateway–3Flag–SRDX have been subjected to considerable research in terms of the function of transcription factors (TFs) via transcription activity and repression, respectively.
Approximately 1500 TFs have been identified in Arabidopsis thaliana genome. To identify their functions, loss-of-function and gain-of-function mutants were generated to analyze the phenotype. However, many loss-of-function mutants did not show any evident phenotype because of the functional redundancy within the TF family. The constitutive misexpression of some TFs may result in lethality or sterility. To overcome these problems, we produced a Gateway-compatible inducible binary vector system that facilitates fast and reliable DNA cloning and biological function identification. The vector can be used for the inducible expression of protein fusions to a polypeptide protein tag named 3xFLAG tag. This vector system can also be used to generate an inducible transcription inhibition of protein fusion to an Ethylene-Responsive Factor-associated amphiphilic repression (EAR) motif. The EAR motif makes it possible to get rid of redundancy within a TF family, thereby facilitating studies on loss of function. With these Gateway vectors, conventional subcloning technology that depends on restriction digestion and ligation is avoided. Thus, these Gateway vectors should be useful not only for the rapid analysis of the functions of redundant plant TFs, but also for the manipulation of TF overexpression, resulting in plant lethality or sterility, via an inducible promoter.
KeywordsTranscriptional activation Transcriptional repression Fusion protein Molecular cloning
We thank Prof. Chuanyou Li (Institute Genetics and Developmental Biology, CAS), Prof. Nam-Hai Chua (The Rockefeller University), and Prof. Detlef Weigel (Max Planck Institute Developmental Biology Germany) for providing the ProLOX:LUC plasmid, pER8 vector, and rTCP4-GFP plasmid, respectively. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31460526, 31760582, and 31400244) and the Natural Science Foundation of Yunnan Province (Grant No. 2017FB050).
- Brand L, Hörler M, Nüesch E, Vassalli S, Barrell P, Yang W, Jefferson RA, Grossniklaus U, Curtis MD (2006) A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol 141(4):1194–1204. https://doi.org/10.1104/pp.106.081299 CrossRefPubMedPubMedCentralGoogle Scholar
- Koyama T, Furutani M, Tasaka M, Ohme-Takagi M (2007) TCP transcription factors control the morphology of shoot lateral organs via negative regulation of the expression of boundary-specific genes in Arabidopsis. Plant Cell 19(2):473–484. https://doi.org/10.1105/tpc.106.044792 CrossRefPubMedPubMedCentralGoogle Scholar
- Qi T, Song S, Ren Q, Wu D, Huang H, Chen Y, Fan M, Peng W, Ren C, Xie D (2011) The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell 23(5):1795–1814. https://doi.org/10.1105/tpc.111.083261 CrossRefPubMedPubMedCentralGoogle Scholar
- Riechmann JL (2002) Transcriptional regulation: a genomic overview. In: Somerville CR, Meyerowitz EM (eds) The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD, p e0085. https://doi.org/10.1199/tab.0085
- Riechmann JL, Heard J, Martin G, Reuber L, Jiang C-Z, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G-L (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290(5499):2105–2110. https://doi.org/10.1126/science.290.5499.2105 CrossRefPubMedGoogle Scholar
- Siligato R, Wang X, Yadav SR, Lehesranta S, Ma G, Ursache R, Sevilem I, Zhang J, Gorte M, Prasad K, Wrzaczek M, Heidstra R, Murphy A, Scheres B, Mähönen AP (2016) MultiSite gateway-compatible cell type-specific gene-inducible system for plants. Plant Physiol 170(2):627–641. https://doi.org/10.1104/pp.15.01246 CrossRefPubMedGoogle Scholar
- Zhai Q, Yan L, Tan D, Chen R, Sun J, Gao L, Dong M-Q, Wang Y, Li C (2013) Phosphorylation-coupled proteolysis of the transcription factor MYC2 is important for jasmonate-signaled plant immunity. PLoS Genet 9(4):e1003422. https://doi.org/10.1371/journal.pgen.1003422 CrossRefPubMedPubMedCentralGoogle Scholar