In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots

Abstract

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules1,2,3, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET–FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET–FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.

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Figure 1: Phasor analysis of in vivo FRET–FLIM.
Figure 2: FRET–FLIM quantification of SCR–SCR, NUC–SCR and SHR–SCR associations in vivo.
Figure 3: FRET–FLIM analysis of JKD–SHR shows interaction in the QC and stem cells.
Figure 4: FRET–FLIM analysis of JKD–SCR shows high interaction in endodermal cells.

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Acknowledgements

We thank P. Benfey for discussions and critical reading of the manuscript, A. Akhmanova and B. P. Bouchet for providing mammalian cells lines and laboratory facilities to perform transfection assays, S. Diaz for sharing DLR protocols used in this study. This work was supported by an NWO VIDI grant to I.B. and Y.L. Y.L. was further supported by ERC Advanced Grant SysArc and NWO Spinoza Grant to B.S. M.P. was supported by an NWO ALW-VIDI grant 864.09.015 and S.W.P. was supported by DFG-project WE 5343/1-1.

Author information

The scientific concept was developed by I.B. and B.S. I.B. and Y.L. designed and executed the experiments. Y.L. generated constructs and lines used for FRET–FLIM experiments, I.B. and Y.L. performed the FRET–FLIM measurement in roots. Y.S., S.W.-P. and R.S. assisted in setting up, optimizing FRET–FLIM devices, data acquisition and analysis in living roots. M.P. performed and quantified the phasor analysis. J.G. helped Y.L. with FRET–FLIM measurements, optimization in protoplast and data analysis and interpretation. T.W.G.J. helped with phasor analysis and discussions related to FRET–FLIM quantification. W.Z. conducted co-immunoprecipitation experiments. Y.L. generated transgenic lines in wild-type and mutant background. I.B. performed DLR luciferase experiments, gene expression analysis by in situ hybridizations, reporter assays and phenotypic analysis in transgenic lines and mutant backgrounds. I.B., Y.L. and B.S. wrote the manuscript. M.-I.S.-P. contributed to HeLa cell experiments by sharing published materials and protocols. I.B., Y.L., B.S., Y.S., S.W.-P., R.S., W.Z., M.P., J.G. and T.W.G.J. contributed to data analysis, interpretation and revision of the manuscript.

Correspondence to Ikram Blilou.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks T. Beeckman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Cell-type organization and co-localization of key transcription factors in the Arabidopsis root meristem and FRET–FLIM determination in Arabidopsis protoplasts.

a, Schematic illustration of the Arabidopsis root meristem, with marked tissue types (cell background colour) and co-localization pattern of SHR, SCR, JKD, NUC and CYCD6;1 (circle colours). The U-shaped domain is encircled by a bold black line. QC, quiescent centre; CEI, cortex/endodermis initial; EN, endodermis; CO, cortex; Vas, vasculature. b, The SHR–SCR–JKD–NUC protein regulatory network (details and references can be found in the text). Red arrow, transcriptional activation; blue flat arrow, transcriptional repression; black line, protein–protein interaction. c, Bimolecular fluorescence complementation assays in Arabidopsis mesophyll protoplasts confirming interaction between SHR, SCR, JKD and NUC (n > 20). d, e, FRET quantification between SHR, SCR, JKD and NUC in protoplasts. Eφ, phase efficiency; Emod, modulation efficiency. Error bars, standard errors (d, e). 28 cells were measured for N-SCR, 64 for SCR-C N-SHR; 53 for SCR-C SHR-C; 97 for N-SHR; 63 for N-SHR E2FA-C; 96 for SHR-C JKD-C; 99 for SHR-C N-JKD; 64 for SCR-C; 101 for SCR-C JKD-C; 95 for SCR-C N-JKD; 93 for SCR-C NUC-C; 66 for SCR-C N-NUC. N and C refer to the position of the fluorophore tagged either to the C terminus (C) or N terminus (N) of each protein. Source Data are available. Source data

Extended Data Figure 2 SHR and SCR complement their respective mutant phenotypes.

a, Image of four-day-old seedlings of wild-type, shr-2, pSHR::SHR:YFP in wild-type, pSHR::YFP:SHR in shr-2 and 35S::SHR:GFP. 20 seedlings per genotype were plated. bg′, Confocal images of three-day-old roots from seedlings of wild type, n = 10 (b, b′); shr-2, n = 11 (c, c′); pSHR:YFP::SHR in wild type, n = 12 (d, d′); pSHR:YFP::SHR in shr-2, n = 11 (e, e′); pSHR::SHR:YFP in wild type, n = 16 (f, f′); and 35S::SHR:GFP, n = 15 (g, g′). h, Image of four-day-old seedlings of wild type, scr-3, pSCR::SCR:YFP in wild type, pSCR::SCR:YFP in scr-3 and 35S::SCR:GFP. 15 seedlings per genotype were plated. im′, Confocal images of three-day-old roots from seedlings of wild type, n = 10 (i, i′); scr-3, n = 10 (j, j′); pSCR::SCR:YFP in wild type, n = 10 (k, k′); pSCR::SCR:YFP in scr-3, n = 11 (l, l′); and 35S::SCR:GFP, n = 15 (m, m′). Insets showing details of tissue layers. co, cortex; en, endodermis; ep, epidermis; mn, monolayer; sn, supernumerary layers; vas, vasculature. Scale bars, 20 μm.

Extended Data Figure 3 Functional analysis of JKD and NUC protein fusions under endogenous and SCR promoters.

a, Image of four-day-old seedlings of wild type, pJKD::JKD:YFP in wild type, pSCR::JKD:YFP in wild type, pSCR::JKD:YFP in jkd-4 and 35S::JKD:YFP. 20 seedlings per genotype were plated. b, Image of four-day-old seedlings of wild type, jkd-4, pJKD::JKD:YFP in wild type, pJKD::JKD:YFP in jkd-4. 15 seedlings per genotype were plated. cj, Confocal images of three-day-old roots from seedlings of wild type, n = 10 (c, c′); jkd-4, n = 12 (d, d′); pJKD::JKD:YFP in wild type, n = 12 (e, e′); pJKD::JKD:YFP in jkd-4, n = 10 (f, f′); pSCR::JKD:YFP in wild type, n = 8 (g, g′); pSCR::JKD:YFP in jkd-4, n = 7 (h, h′); wild type, n = 7 (i) and 35S::JKD:YFP, n = 14 (j). Insets showing details of tissue layers. k, Image of four-day-old seedlings of wild type; pNUC::NUC:YFP in wild type; pSCR::NUC:YFP in wild type and 35S::NUC:YFP. 20 seedlings per genotype were plated. lo, Confocal images of three-day-old roots from seedlings of pNUC::NUC:YFP in wild type, n = 10 (l, l′); pSCR::NUC:YFP in wild type, n = 10 (m, m′); wild type, n = 10 (n); and 35S::NUC:mCherry, n = 24 (o). Insets show details of tissue layers. co, cortex; en, endodermis; ep, epidermis; pe, pericycle; vas, vasculature. Arrow points to elongated cells exiting the meristem. Scale bars, 20 μm.

Extended Data Figure 4 Analysis of fluorescence protein intensity under endogenous promoters.

ad, Confocal images of three-day-old roots from seedlings of pSCR::SCR:YFP, pSHR:SHR:YFP, pJKD::JKD:YFP and pNUC::NUC:YFP. Scale bar, 20 μm. e, Box plots showing differences in YFP intensity (16-bit) of roots expressing endogenous fusions of SCR (n = 83), SHR (n = 86), JKD (n = 62) and NUC (n = 24). n represents the number of measured nuclei. The box plot displays the distribution of fluorescence intensity: median, 1st and 3rd quartiles, minimum and maximum. The error bars indicate minimum and maximum of distribution.

Extended Data Figure 5 Panels of donor fluorescence lifetime and fluorescence intensity of YFP and RFP channels of all protein pairs used in this study.

All heat maps are the same ones used in Figs 2, 3, 4 with the same colour scale. YFP and RFP channel intensity images of the same roots are next to the heat maps displayed in Figs 2, 3, 4. Number of technical and biological replicates can be found in Supplementary Tables 1–5.

Extended Data Figure 6 JKD amplifies WOX5 activity in the vasculature.

af, Confocal images of embryos of wild type (ac) and pWOX5::JKD:YFP stained with mPS-PI. (df). a, b, n = 13; c, n = 10; d, e, n = 35; f, n = 22. gi, Confocal images of embryos expressing pWOX5::CFP, n = 14. jl, Confocal images of embryos expressing pWOX5::JKD:YFP, n = 15. Scale bars, 10 μm (aj, k), 50 μm (l). mn′, Confocal images of roots of expressing pWOX5::CFP in wild type, n = 20 (m, m′) and pWOX5::JKD:YFP,. n = 14 (n, n′). o, p, Confocal images of roots of wild type, n = 10 (o) and pWOX5::JKD:YFP, n = 8 stained with mPS-PI (p). q, r, In situ hybridization showing WOX5 mRNA expression in wild type, n = 27 (q) and pWOX5::JKD:YFP, n = 29 (r). Scale bars, 20 μm (mr).

Extended Data Figure 7 JKD expression in the vasculature does not affect WOX5 activity.

a, b, Confocal images of roots of wild type, n = 10 (a) and pWOL>>JKD:mCherry, n = 13 (b) stained with mPS-PI. Scale bars, 10 μm. c, d, In situ hybridization showing WOX5 mRNA expression in wild type, n = 20 (c) and pWOL>>JKD:mCherry, n = 32 (d). Scale bars, 20 μm. ef′, Confocal images of roots expressing pWOX5::CFP in wild type, n = 22 (e, e′) and in pWOL>>JKD:mCherry, n = 27 (f, f′). Scale bars, 10 μm.

Extended Data Figure 8 SHR, SCR and JKD form ternary complex.

ad, Protein binding competition and co-association tested by split-LUC assay in HeLa cells. HeLa cells experiments were performed in triplicates and repeated three times. Error bars indicate standard deviations. e, Co-immunoprecipitation of SHR, SCR and JKD from transfected Nicotiana benthamiana leaves. CoiP experiments for SCR, SHR and JKD were done seven times; experiments including negative controls were done two times. Full scan gels of one experiment can be found in the Supplementary Information.

Extended Data Figure 9 Spatial network distribution specifying distinct cell types.

Scheme representing the spatial protein complex distribution and the cellular sub-networks in the QC, CEI, meristemic endodermis and mature endodermis. SHR moves from the vasculature to the U-shaped domain where a close association with JKD in the QC leads to WOX5 expression and QC specification, a close association to SCR in the CEI activates CYCD6;1 and promote formative divisions, while its association to closely conjoint SCR and JKD leads to endodermal specification.

Extended Data Table 1 FRET quantifications displayed as intensity based relative to: the positive control (upper rows), the maximal FRET state (DA) (middle rows) and the estimated Förster resonance energy transfer (E) (lower rows)

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Long, Y., Stahl, Y., Weidtkamp-Peters, S. et al. In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots. Nature 548, 97–102 (2017). https://doi.org/10.1038/nature23317

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