Abstract
Phytochromes are a family of photoreceptors in plants that perceive the red (R) and far-red (FR) components of their light environment. Phytochromes exist in vivo in two forms, the inactive Pr form and the active Pfr form, that are interconvertible by treatments with R or FR light. It is believed that phytochromes transduce light signals by interacting with their signaling partners. A GAL4-based light-switchable yeast two-hybrid (Y2H) system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components. However, several pairs of interactions between phytochromes and their interactors, such as the phyA-COP1 and phyA-TZP interactions, were demonstrated by other assay systems but were not detected by this GAL4 Y2H system. Here, we report a modified LexA Y2H system, in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein. The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin (PCB) extracted from Spirulina. The well-defined interaction pairs, including phyA-FHY1 and phyB-PIFs, are well reproducible in this system. Moreover, we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions. Together, our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.
Similar content being viewed by others
References
Bae G, Choi G (2008) Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol 59:281–311. https://doi.org/10.1146/annurev.arplant.59.032607.092859
Bu Q, Castillon A, Chen F, Zhu L, Huq E (2011) Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis. Plant Mol Biol 77:501–511. https://doi.org/10.1007/s11103-011-9827-4
Burgie ES, Vierstra RD (2014) Phytochromes: an atomic perspective on photoactivation and signaling. Plant Cell 26:4568–4583. https://doi.org/10.1105/tpc.114.131623
Castillon A, Shen H, Huq E (2009) Blue light induces degradation of the negative regulator phytochrome interacting factor 1 to promote photomorphogenic development of Arabidopsis seedlings. Genetics 182:161–171. https://doi.org/10.1534/genetics.108.099887
Dalton JC, Batz U, Liu J, Curie GL, Quail PH (2016) A modified reverse one-hybrid screen identifies transcriptional activation domains in PHYTOCHROME-INTERACTING FACTOR 3. Front Plant Sci 7:881. https://doi.org/10.3389/fpls.2016.00881
Dong J, Tang D, Gao Z, Yu R, Li K, He H, Terzaghi W, Deng XW, Chen H (2014) Arabidopsis DE-ETIOLATED1 represses photomorphogenesis by positively regulating phytochrome-interacting factors in the dark. Plant Cell 26:3630–3645. https://doi.org/10.1105/tpc.114.130666
Dong X, Yan Y, Jiang B, Shi Y, Jia Y, Cheng J, Shi Y, Kang J, Li H, Zhang D, Qi L, Han R, Zhang S, Zhou Y, Wang X, Terzaghi W, Gu H, Kang D, Yang S, Li J (2020) The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures. EMBO J 39:e103630. https://doi.org/10.15252/embj.2019103630
Enderle B, Sheerin DJ, Paik I, Kathare PK, Schwenk P, Klose C, Ulbrich MH, Huq E, Hiltbrunner A (2017) PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion. Nat Commun 8:2221. https://doi.org/10.1038/s41467-017-02311-8
Ferro E, Trabalzini L (2013) The yeast two-hybrid and related methods as powerful tools to study plant cell signalling. Plant Mol Biol 83:287–301. https://doi.org/10.1007/s11103-013-0094-4
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246. https://doi.org/10.1038/340245a0
Hiltbrunner A, Viczian A, Bury E, Tscheuschler A, Kircher S, Toth R, Honsberger A, Nagy F, Fankhauser C, Schafer E (2005) Nuclear accumulation of the phytochrome A photoreceptor requires FHY1. Curr Biol 15:2125–2130. https://doi.org/10.1016/j.cub.2005.10.042
Hiltbrunner A, Tscheuschler A, Viczian A, Kunkel T, Kircher S, Schafer E (2006) FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor. Plant Cell Physiol 47:1023–1034. https://doi.org/10.1093/pcp/pcj087
Holm M, Hardtke CS, Gaudet R, Deng XW (2001) Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1. EMBO J 20:118–127. https://doi.org/10.1093/emboj/20.1.118
Hughes RM, Vrana JD, Song J, Tucker CL (2012) Light-dependent, dark-promoted interaction between Arabidopsis cryptochrome 1 and phytochrome B proteins. J Biol Chem 287:22165–22172. https://doi.org/10.1074/jbc.M112.360545
Jiao Y, Lau OS, Deng XW (2007) Light-regulated transcriptional networks in higher plants. Nat Rev Genet 8:217–230. https://doi.org/10.1038/nrg2049
Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods 7:973–975. https://doi.org/10.1038/nmeth.1524
Khanna R, Huq E, Kikis EA, Al-Sady B, Lanzatella C, Quail PH (2004) A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. Plant Cell 16:3033–3044. https://doi.org/10.1105/tpc.104.025643
Kikis EA, Oka Y, Hudson ME, Nagatani A, Quail PH (2009) Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. PLoS Genet 5:e1000352. https://doi.org/10.1371/journal.pgen.1000352
Lagarias JC, Mercurio FM (1985) Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro. J Biol Chem 260:2415–2423
Lee N, Choi G (2017) Phytochrome-interacting factor from Arabidopsis to liverwort. Curr Opin Plant Biol 35:54–60. https://doi.org/10.1016/j.pbi.2016.11.004
Legris M, Ince YC, Fankhauser C (2019) Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nat Commun 10:5219. https://doi.org/10.1038/s41467-019-13045-0
Leivar P, Quail PH (2011) PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16:19–28. https://doi.org/10.1016/j.tplants.2010.08.003
Leivar P, Monte E, Al-Sady B, Carle C, Storer A, Alonso JM, Ecker JR, Quail PH (2008) The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged red light by modulating phyB levels. Plant Cell 20:337–352. https://doi.org/10.1105/tpc.107.052142
Levskaya A, Weiner OD, Lim WA, Voigt CA (2009) Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461:997–1001. https://doi.org/10.1038/nature08446
Li J, Li G, Gao S, Martinez C, He G, Zhou Z, Huang X, Lee JH, Zhang H, Shen Y, Wang H, Deng XW (2010) Arabidopsis transcription factor ELONGATED HYPOCOTYL5 plays a role in the feedback regulation of phytochrome A signaling. Plant Cell 22:3634–3649. https://doi.org/10.1105/tpc.110.075788
Li J, Li G, Wang H, Deng XW (2011) Phytochrome signaling mechanisms. Arabidopsis Book 9:e0148. https://doi.org/10.1199/tab.0148
Lu XD, Zhou CM, Xu PB, Luo Q, Lian HL, Yang HQ (2015) Red-light-dependent interaction of phyB with SPA1 promotes COP1-SPA1 dissociation and photomorphogenic development in Arabidopsis. Mol Plant 8:467–478. https://doi.org/10.1016/j.molp.2014.11.025
Miesenbock G (2011) Optogenetic control of cells and circuits. Annu Rev Cell Dev Biol 27:731–758. https://doi.org/10.1146/annurev-cellbio-100109-104051
Ni M, Tepperman JM, Quail PH (1998) PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein. Cell 95:657–667. https://doi.org/10.1016/s0092-8674(00)81636-0
Oka Y, Matsushita T, Mochizuki N, Suzuki T, Tokutomi S, Nagatani A (2004) Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis. Plant Cell 16:2104–2116. https://doi.org/10.1105/tpc.104.022350
Pham VN, Kathare PK, Huq E (2018) Phytochromes and phytochrome interacting factors. Plant Physiol 176:1025–1038. https://doi.org/10.1104/pp.17.01384
Possart A, Hiltbrunner A (2013) An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants. Plant Cell 25:102–114. https://doi.org/10.1105/tpc.112.104331
Qi L, Liu S, Li C, Fu J, Jing Y, Cheng J, Li H, Zhang D, Wang X, Dong X, Han R, Li B, Zhang Y, Li Z, Terzaghi W, Song C-P, Lin R, Gong Z, Li J (2020) PHYTOCHROME-INTERACTING FACTORS interact with the ABA receptors PYL8 and PYL9 to orchestrate ABA signaling in darkness. Mol Plant 13:414–430. https://doi.org/10.1016/j.molp.2020.02.001
Rausenberger J, Tscheuschler A, Nordmeier W, Wust F, Timmer J, Schafer E, Fleck C, Hiltbrunner A (2011) Photoconversion and nuclear trafficking cycles determine phytochrome A’s response profile to far-red light. Cell 146:813–825. https://doi.org/10.1016/j.cell.2011.07.023
Repina NA, Rosenbloom A, Mukherjee A, Schaffer DV, Kane RS (2017) At light speed: advances in optogenetic systems for regulating cell signaling and behavior. Annu Rev Chem Biomol Eng 8:13–39. https://doi.org/10.1146/annurev-chembioeng-060816-101254
Rockwell NC, Su YS, Lagarias JC (2006) Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 57:837–858. https://doi.org/10.1146/annurev.arplant.56.032604.144208
Saijo Y, Zhu D, Li J, Rubio V, Zhou Z, Shen Y, Hoecker U, Wang H, Deng XW (2008) Arabidopsis COP1/SPA1 complex and FHY1/FHY3 associate with distinct phosphorylated forms of phytochrome A in balancing light signaling. Mol Cell 31:607–613. https://doi.org/10.1016/j.molcel.2008.08.003
Seo HS, Watanabe E, Tokutomi S, Nagatani A, Chua NH (2004) Photoreceptor ubiquitination by COP1 E3 ligase desensitizes phytochrome A signaling. Genes Dev 18:617–622. https://doi.org/10.1101/gad.1187804
Sheerin DJ (2019) Investigation of light-regulated protein-protein interactions using yeast two-hybrid assays. In: Hiltbrunner A (ed) Phytochromes: methods and protocols. Springer New York, New York, pp 1–19. https://doi.org/10.1007/978-1-4939-9612-4_1
Sheerin DJ, Menon C, zur Oven-Krockhaus S, Enderle B, Zhu L, Johnen P, Schleifenbaum F, Stierhof YD, Huq E, Hiltbrunner A (2015) Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell 27:189–201. https://doi.org/10.1105/tpc.114.134775
Shen Y, Khanna R, Carle CM, Quail PH (2007) Phytochrome induces rapid PIF5 phosphorylation and degradation in response to red-light activation. Plant Physiol 145:1043–1051. https://doi.org/10.1104/pp.107.105601
Shen Y, Zhou Z, Feng S, Li J, Tan-Wilson A, Qu LJ, Wang H, Deng XW (2009) Phytochrome A mediates rapid red light-induced phosphorylation of Arabidopsis FAR-RED ELONGATED HYPOCOTYL1 in a low fluence response. Plant Cell 21:494–506. https://doi.org/10.1105/tpc.108.061259
Shimizu-Sato S, Huq E, Tepperman JM, Quail PH (2002) A light-switchable gene promoter system. Nat Biotechnol 20:1041–1044. https://doi.org/10.1038/nbt734
Sorokina O, Kapus A, Terecskei K, Dixon LE, Kozma-Bognar L, Nagy F, Millar AJ (2009) A switchable light-input, light-output system modelled and constructed in yeast. J Biol Eng 3:15. https://doi.org/10.1186/1754-1611-3-15
Su YS, Lagarias JC (2007) Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants. Plant Cell 19:2124–2139. https://doi.org/10.1105/tpc.107.051516
Torii KU, McNellis TW, Deng XW (1998) Functional dissection of Arabidopsis COP1 reveals specific roles of its three structural modules in light control of seedling development. EMBO J 17:5577–5587. https://doi.org/10.1093/emboj/17.19.5577
Trupkin SA, Debrieux D, Hiltbrunner A, Fankhauser C, Casal JJ (2007) The serine-rich N-terminal region of Arabidopsis phytochrome A is required for protein stability. Plant Mol Biol 63:669–678. https://doi.org/10.1007/s11103-006-9115-x
Viczian A, Adam E, Wolf I, Bindics J, Kircher S, Heijde M, Ulm R, Schafer E, Nagy F (2012) A short amino-terminal part of Arabidopsis phytochrome A induces constitutive photomorphogenic response. Mol Plant 5:629–641. https://doi.org/10.1093/mp/sss035
Vidal M, Legrain P (1999) Yeast forward and reverse ’n’-hybrid systems. Nucleic Acids Res 27:919–929. https://doi.org/10.1093/nar/27.4.919
Xin R, Kathare PK, Huq E (2019) Coordinated regulation of pre-mRNA splicing by the SFPS-RRC1 complex to promote photomorphogenesis. Plant Cell 31:2052–2069. https://doi.org/10.1105/tpc.18.00786
Xu X, Paik I, Zhu L, Huq E (2015) Illuminating progress in phytochrome-mediated light signaling pathways. Trends Plant Sci 20:641–650. https://doi.org/10.1016/j.tplants.2015.06.010
Yan Y, Li C, Dong X, Li H, Zhang D, Zhou Y, Jiang B, Peng J, Qin X, Cheng J, Wang X, Song P, Qi L, Zheng Y, Li B, Terzaghi W, Yang S, Guo Y, Li J (2020) MYB30 is a key negative regulator of Arabidopsis photomorphogenic development that promotes PIF4 and PIF5 protein accumulation in the light. Plant Cell 32:2196–2215. https://doi.org/10.1105/tpc.19.00645
Yang SW, Jang IC, Henriques R, Chua NH (2009) FAR-RED ELONGATED HYPOCOTYL1 and FHY1-LIKE associate with the Arabidopsis transcription factors LAF1 and HFR1 to transmit phytochrome A signals for inhibition of hypocotyl elongation. Plant Cell 21:1341–1359. https://doi.org/10.1105/tpc.109.067215
Yang X, Jost AP, Weiner OD, Tang C (2013) A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast. Mol Biol Cell 24:2419–2430. https://doi.org/10.1091/mbc.E13-03-0126
Yazawa M, Sadaghiani AM, Hsueh B, Dolmetsch RE (2009) Induction of protein-protein interactions in live cells using light. Nat Biotechnol 27:941–945. https://doi.org/10.1038/nbt.1569
Zhang S, Li C, Zhou Y, Wang X, Li H, Feng Z, Chen H, Qin G, Jin D, Terzaghi W, Gu H, Qu LJ, Kang D, Deng XW, Li J (2018) TANDEM ZINC-FINGER/PLUS3 is a key component of phytochrome A signaling. Plant Cell 30:835–852. https://doi.org/10.1105/tpc.17.00677
Zhou Y, Yang L, Duan J, Cheng J, Shen Y, Wang X, Han R, Li H, Li Z, Wang L, Terzaghi W, Zhu D, Chen H, Deng XW, Li J (2018) Hinge region of Arabidopsis phyA plays an important role in regulating phyA function. Proc Natl Acad Sci USA 115:E11864–E11873. https://doi.org/10.1073/pnas.1813162115
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (31970262 and 31770321), Beijing Outstanding University Discipline Program, and the Recruitment Program of Global Youth Experts of China.
Author information
Authors and Affiliations
Contributions
HL and JL designed research. HL, XQ, PS, and RH performed research. JL and HL analyzed the data. JL and HL wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Li, H., Qin, X., Song, P. et al. A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners. aBIOTECH 2, 105–116 (2021). https://doi.org/10.1007/s42994-021-00034-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42994-021-00034-5