Модификация оптогенетической системы BphP1-QPAS1 для регуляции экспрессии генов в листьях табака Nicotiana benthamiana с помощью ближнего инфракрасного света

В растениях регуляция транскрипции трансгенов обычно осуществляется с помощью химических агентов. Безопасной альтернативой химически индуцируемым системам могут служить оптогенетические (т. е. светоиндуцируемые) системы. Бактериальная система BphP1-QPAS1 выгодно отличается от других оптогенетических систем, поскольку активируется ближним инфракрасным светом (БИК, 780 нм), выходящим за пределы спектра восприятия растительных фоторецепторов. Система BphP1-QPAS1 основана на использовании «расщепленного» транскрипционного фактора (ТФ), состоящего из ДНК-связывающего и димеризующего доменов дрожжевого ТФ Gal4, слитого с компонентом QPAS1, и трансактивационного домена VP16, слитого с BphP1. Под действием БИК света BphP1 взаимодействует с QPAS1, что приводит к сближению Gal4 с VP16 и запуску экспрессии репортерного гена. Основным препятствием для широкого использования оптогенетических систем в растениях является нежелательная активация таких систем при воздействии дневного (белого) света, который включает широкий спектр длин волн и жизненно важен для нормального роста растений. Решением проблемы нежелательной активации может стать временное удаление из ядра клетки одного из компонентов «расщепленного» ТФ при воздействии белого света. В данной работе мы модифицировали систему BphP1-QPAS1 для активации экспрессии репортерного гена в листьях табака Nicotiana benthamiana с помощью БИК света. Для этого мы комбинировали систему BphP1-QPAS1 с несколькими вариантами LOV домен-содержащих белков, активируемых синим светом (460–480 нм). Наилучшие результаты были достигнуты при комбинации компонентов системы BphP1-QPAS1 с доменом AsLOV2 из белка фототропин 1 Avena sativa, несущим на С концевой Jα спирали последовательность дегрона RRRG и запускающим деградацию химерного белка NES-Gal4-QPAS1-AsLOV2-RRRG при воздействии белого света. Такая модификация активировала систему BphP1-QPAS1 в листьях табака только при воздействии БИК света, но не в темноте либо при белом свете. Мы полагаем, что в будущем система BphP1-QPAS1 может быть применена для повышения устойчивости растений к неблагоприятным условиям среды, вредителям, вирусным заболеваниям.

1. Andres J., Blomeier T., Zurbriggen M.D. Synthetic switches and regulatory circuits in plants. Plant Physiol. 2019;179(3):862-884. doi 10.1104/pp.18.01362

2. Auldridge M.E., Forest K.T. Bacterial phytochromes: more than meets the light. Crit Rev Biochem Mol Biol. 2011;46(1):67-88. doi 10.3109/10409238.2010.546389

3. Battle M.W., Jones M.A. Cryptochromes integrate green light signals into the circadian system. Plant Cell Environ. 2020;43(1):16-27. doi 10.1111/pce.13643

4. Bischof J., Maeda R.K., Hediger M., Karch F., Basler K. An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases. Proc Natl Acad Sci USA. 2007;104(9):3312-3317. doi 10.1073/pnas.0611511104

5. Bonger K.M., Rakhit R., Payumo A.Y., Chen J.K., Wandless T.J. General method for regulating protein stability with light. ACS Chem Biol. 2014;9(1):111-115. doi 10.1021/cb400755b

6. Braguy J., Zurbriggen M.D. Synthetic strategies for plant signalling studies: molecular toolbox and orthogonal platforms. Plant J. 2016; 87(1):118-138. doi 10.1111/tpj.13218

7. Chatelle C., Ochoa-Fernandez R., Engesser R., Schneider N., Beyer H.M., Jones A.R., Timmer J., Zurbriggen M.D., Weber W. A green-light-responsive system for the control of transgene expression in mammalian and plant cells. ACS Synth Biol. 2018;7(5): 1349-1358. doi 10.1021/acssynbio.7b00450

8. Chen P.-Y., Wang C.-K., Soong S.-C., To K.-Y. Complete sequence of the binary vector pBI121 and its application in cloning T-DNA in sertion from transgenic plants. Mol Breed. 2003;11(4):287-293. doi 10.1023/A:1023475710642

9. Chernov K.G., Redchuk T.A., Omelina E.S., Verkhusha V.V. Near-infrared fluorescent proteins, biosensors, and optogenetic tools engineered from phytochromes. Chem Rev. 2017;117(9):6423-6446. doi 10.1021/acs.chemrev.6b00700

10. Christie J.M., Zurbriggen M.D. Optogenetics in plants. New Phytol. 2021;229(6):3108-3115. doi 10.1111/nph.17008

11. Christie J.M., Gawthorne J., Young G., Fraser N.J., Roe A.J. LOV to BLUF: flavoprotein contributions to the optogenetic toolkit. Mol Plant. 2012;5(3):533-544. doi 10.1093/mp/sss020

12. Crosson S., Moffat K. Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction. Proc Natl Acad Sci USA. 2001;98(6):2995-3000. doi 10.1073/pnas.051520298

13. Gälweiler L., Conlan R.S., Mader P., Palme K., Moore I. Technical advance: the DNA-binding activity of Gal4 is inhibited by methylation of the Gal4 binding site in plant chromatin. Plant J. 2000;23(1):143- 157. doi 10.1046/j.1365-313x.2000.00805.x

14. Gayatri T., Basu A. Development of reproducible regeneration and transformation system for Sesamum indicum. Plant Cell Tiss Organ Cult. 2020;143(2):441-456. doi 10.1007/s11240-020-01931-1

15. Giraud E., Verméglio A. Bacteriophytochromes in anoxygenic photosynthetic bacteria. Photosynth Res. 2008;97(2):141-153. doi 10.1007/s11120-008-9323-0

16. Gligorovski V., Sadeghi A., Rahi S.J. Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor. Nat Commun. 2023;14(1):3810. doi 10.1038/s41467-023-38959-8

17. Guidi L., Tattini M., Landi M. How does chloroplast protect chlorophyll against excessive light? In: Chlorophyll. InTech, 2017. doi 10.5772/67887

18. Hernández-Candia C.N., Tucker C.L. Optogenetic control of gene expression using cryptochrome 2 and a light-activated degron. Methods Mol Biol. 2020;2173:151-158. doi 10.1007/978-1-0716-0755-8_10

19. Herrou J., Crosson S. Function, structure and mechanism of bacterial photosensory LOV proteins. Nat Rev Microbiol. 2011;9(10):713- 723. doi 10.1038/nrmicro2622

20. Johnson A.A.T., Hibberd J.M., Gay C., Essah P.A., Haseloff J., Tester M., Guiderdoni E. Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system. Plant J. 2005;41(5):779-789. doi 10.1111/j.1365-313X.2005.02339.x

21. Johnson M.P. Photosynthesis. Essays Biochem. 2016;60(3):255-273. doi 10.1042/EBC20160016

22. Kaberniuk A.A., Shemetov A.A., Verkhusha V.V. A bacterial phytochrome-based optogenetic system controllable with near-infrared light. Nat Methods. 2016;13(7):591-597. doi 10.1038/nmeth.3864

23. Kaya S.G., Hovan A., Fraaije M.W. Engineering of LOV-domains for their use as protein tags. Arch Biochem Biophys. 2025;763:110228. doi 10.1016/j.abb.2024.110228

24. Kim C., Yun S.R., Lee S.J., Kim S.O., Lee H., Choi J., Kim J.G., Kim T.W., You S., Kosheleva I., Noh T., Baek J., Ihee H. Structural dynamics of protein-protein association involved in the lightinduced transition of Avena sativa LOV2 protein. Nat Commun. 2024;15(1):6991. doi 10.1038/s41467-024-51461-z

25. Konermann S., Brigham M.D., Trevino A.E., Hsu P.D., Heidenreich M., Cong L., Platt R.J., Scott D.A., Church G.M., Zhang F. Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013;500(7463):472-476. doi 10.1038/nature12466

26. Krueger D., Izquierdo E., Viswanathan R., Hartmann J., Pallares Cartes C., De Renzis S. Principles and applications of optogenetics in developmental biology. Development. 2019;146(20):dev175067. doi 10.1242/dev.175067

27. Li F.W., Melkonian M., Rothfels C.J., Villarreal J.C., Stevenson D.W., Graham S.W., Wong G.K., Pryer K.M., Mathews S. Phytochrome diversity in green plants and the origin of canonical plant phytochromes. Nat Commun. 2015;6(1):7852. doi 10.1038/ncomms8852

28. Li J., Li G., Wang H., Wang Deng X. Phytochrome signaling mechanisms. Arabidopsis Book. 2011;9:e0148. doi 10.1199/tab.0148

29. Losi A., Gärtner W. Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors. Photochem Photobiol. 2011;87(3):491-510. doi 10.1111/j.1751-1097.2011.00913.x

30. Lungu O.I., Hallett R.A., Choi E.J., Aiken M.J., Hahn K.M., Kuhlman B. Designing photoswitchable peptides using the AsLOV2 domain. Chem Biol. 2012;19(4):507-517. doi 10.1016/j.chembiol.2012.02.006

31. Mathews S. Evolutionary studies illuminate the structural-functional model of plant phytochromes. Plant Cell. 2010;22(1):4-16. doi 10.1105/tpc.109.072280

32. Müller K., Engesser R., Metzger S., Schulz S., Kämpf M.M., Busacker M., Steinberg T., Tomakidi P., Ehrbar M., Nagy F., Timmer J., Zubriggen M.D., Weber W. A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells. Nucleic Acids Res. 2013;41(7):e77. doi 10.1093/nar/gkt002

33. Müller K., Siegel D., Rodriguez Jahnke F., Gerrer K., Wend S., Decker E.L., Reski R., Weber W., Zurbriggen M.D. A red light-controlled synthetic gene expression switch for plant systems. Mol Biosyst. 2014;10(7):1679-1688. doi 10.1039/c3mb70579j

34. Müller K., Zurbriggen M.D., Weber W. An optogenetic upgrade for the Tet‐OFF system. Biotechnol Bioeng. 2015;112(7):1483-1487. doi 10.1002/bit.25562

35. Ni M., Tepperman J.M., Quail P.H. Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature. 1999;400(6746):781-784. doi 10.1038/23500

36. Niopek D., Wehler P., Roensch J., Eils R., Di Ventura B. Optogenetic control of nuclear protein export. Nat Commun. 2016;7:10624. doi 10.1038/ncomms10624

37. Noda N., Ozawa T. Light-controllable transcription system by nucleocytoplasmic shuttling of a truncated phytochrome B. Photochem Photobiol. 2018;94(5):1071-1076. doi 10.1111/php.12955

38. Ochoa-Fernandez R., Samodelov S.L., Brandl S.M., Wehinger E., Müller K., Weber W., Zurbriggen M.D. Optogenetics in plants: red/farred light control of gene expression. Methods Mol Biol. 2016;1408: 125-139. doi 10.1007/978-1-4939-3512-3_9

39. Ochoa-Fernandez R., Abel N.B., Wieland F.-G., Schlegel J., Koch L.A., Miller J.B., Engesser R., Giuriani G., Brandl S.M., Timmer J., Weber W., Ott T., Simon R., Zurbriggen M.D. Optogenetic control of gene expression in plants in the presence of ambient white light. Nat Methods. 2020;17(7):717-725. doi 10.1038/s41592-020-0868-y

40. Omelina E.S., Pindyurin A.V. Optogenetic regulation of endogenous gene transcription in mammals. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2019;23(2):219-225. doi 10.18699/VJ19.485

41. Omelina E.S., Yushkova A.A., Motorina D.M., Volegov G.A., Kozhevnikova E.N., Pindyurin A.V. Optogenetic and chemical induction systems for regulation of transgene expression in plants: use in basic and applied research. Int J Mol Sci. 2022;23(3):1737. doi 10.3390/ijms23031737

42. Pathak G.P., Spiltoir J.I., Höglund C., Polstein L.R., Heine-Koskinen S., Gersbach C.A., Rossi J., Tucker C.L. Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. Nucleic Acids Res. 2017;45(20):e167. doi 10.1093/nar/gkx260

43. Peter E., Dick B., Baeurle S.A. Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa. Nat Commun. 2010;1:122. doi 10.1038/ncomms1121

44. Piatkevich K.D., Subach F.V., Verkhusha V.V. Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals. Chem Soc Rev. 2013;42(8):3441-3452. doi 10.1039/c3cs35458j

45. Qian Y., Li T., Zhou S., Chen X., Yang Y. A single-component optogenetic Gal4-UAS system allows stringent control of gene expression in zebrafish and Drosophila. ACS Synth Biol. 2023;12(3):664-671. doi 10.1021/acssynbio.2c00410

46. Redchuk T.A., Omelina E.S., Chernov K.G., Verkhusha V.V. Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nat Chem Biol. 2017;13(6):633-639. doi 10.1038/nchembio.2343

47. Redchuk T.A., Karasev M.M., Omelina E.S., Verkhusha V.V. Nearinfrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells. Chembiochem. 2018;19(12):1334- 1340. doi 10.1002/cbic.201700642

48. Renicke C., Schuster D., Usherenko S., Essen L.O., Taxis C. A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chem Biol. 2013;20(4):619-626. doi 10.1016/j.chembiol.2013.03.005

49. Sakvarelidze L., Tao Z., Bush M., Roberts G.R., Leader D.J., Doonan J.H., Rawsthorne S. Coupling the GAL4 UAS system with alcR for versatile cell type‐specific chemically inducible gene expression in Arabidopsis. Plant Biotechnol J. 2007;5(4):465-476. doi 10.1111/j.1467-7652.2007.00254.x

50. Shcherbakova D.M., Shemetov A.A., Kaberniuk A.A., Verkhusha V.V. Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu Rev Biochem. 2015;84(1):519-550. doi 10.1146/annurev-biochem-060614-034411

51. Shikata H., Denninger P. Plant optogenetics: applications and perspectives. Curr Opin Plant Biol. 2022;68:102256. doi 10.1016/j.pbi.2022.102256

52. Shimizu-Sato S., Huq E., Tepperman J.M., Quail P.H. A light-switchable gene promoter system. Nat Biotechnol. 2002;20(10):1041-1044. doi 10.1038/nbt734

53. Strickland D., Lin Y., Wagner E., Hope C.M., Zayner J., Antoniou C., Sosnick T.R., Weiss E.L., Glotzer M. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods. 2012; 9(4):379-384. doi 10.1038/nmeth.1904

54. Wagner J.R., Zhang J., Brunzelle J.S., Vierstra R.D., Forest K.T. High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution. J Biol Chem. 2007;282(16):12298-12309. doi 10.1074/jbc.M611824200

55. Wang X., Chen X., Yang Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat Methods. 2012;9(3): 266-269. doi 10.1038/nmeth.1892

56. Willumsen B.M., Christensen A., Hubbert N.L., Papageorge A.G., Lowy D.R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984a;310(5978):583-586. doi 10.1038/310583a0

57. Willumsen B.M., Norris K., Papageorge A.G., Hubbert N.L., Lowy D.R. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984b;3(11):2581-2585. doi 10.1002/j.1460-2075.1984.tb02177.x

58. Wu C., Li X., Yuan W., Chen G., Kilian A., Li J., Xu C., Li X., Zhou D.X., Wang S., Zhang Q. Development of enhancer trap lines for functional analysis of the rice genome. Plant J. 2003;35(3):418- 427. doi 10.1046/j.1365-313x.2003.01808.x

59. Wu Y.I., Frey D., Lungu O.I., Jaehrig A., Schlichting I., Kuhlman B., Hahn K.M. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature. 2009;461(7260):104-108. doi 10.1038/nature08241

60. Yang X., Kuk J., Moffat K. Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction. Proc Natl Acad Sci USA. 2008;105(38):14715-14720. doi 10.1073/pnas.0806718105

61. Yumerefendi H., Dickinson D.J., Wang H., Zimmerman S.P., Bear J.E., Goldstein B., Hahn K., Kuhlman B. Control of protein activity and cell fate specification via light-mediated nuclear translocation. PLoS One. 2015;10(6):e0128443. doi 10.1371/journal.pone.0128443

62. Yun A., Kang J., Lee J., Song S.J., Hwang I. Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana). Front Plant Sci. 2023;14:1138089. doi 10.3389/fpls.2023.1138089

63. Zayner J.P., Antoniou C., Sosnick T.R. The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J Mol Biol. 2012;419(1-2):61-74. doi 10.1016/j.jmb.2012.02.037

64. Zheng X., Deng W., Luo K., Duan H., Chen Y., McAvoy R., Song S., Pei Y., Li Y. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep. 2007;26(8):1195- 1203. doi 10.1007/s00299-007-0307-x

65. Zhu Y., Tepperman J.M., Fairchild C.D., Quail P.H. Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3. Proc Natl Acad Sci USA. 2000;97(24):13419-13424. doi 10.1073/pnas.230433797

66. Zoltowski B.D., Crane B.R. Light activation of the LOV protein vivid generates a rapidly exchanging dimer. Biochemistry. 2008; 47(27):7012-7019. doi 10.1021/bi8007017

67. Zoltowski B.D., Schwerdtfeger C., Widom J., Loros J.J., Bilwes A.M., Dunlap J.C., Crane B.R. Conformational switching in the fungal light sensor Vivid. Science. 2007;316(5827):1054-1057. doi 10.1126/science.1137128