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Minimum requirements for changing and maintaining endodermis cell identity in the Arabidopsis root

Abstract

Changes in gene regulation during differentiation are governed by networks of transcription factors. The Arabidopsis root endodermis is a tractable model to address how transcription factors contribute to differentiation. We used a bottom-up approach to understand the extent to which transcription factors that are required for endodermis differentiation can confer endodermis identity to a non-native cell type. Our results show that the transcription factors SHORTROOT and MYB36 alone have limited ability to induce ectopic endodermal features in the absence of additional cues. The stele-derived signalling peptide CIF2 stabilizes SHORTROOT-induced endodermis identity acquisition. The outcome is a partially impermeable barrier deposited in the subepidermal cell layer, which has a transcriptional signature similar to the endodermis. These results demonstrate that other root cell types can be forced to differentiate into the endodermis and highlight a previously unappreciated role for receptor kinase signalling in maintaining endodermis identity.

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Fig. 1: CIF2 stabilizes transcription factor-induced endodermis identity acquisition.
Fig. 2: The identity of the subepidermis before and after CIF2 treatment.
Fig. 3: Dynamics of ectopic CASP1 expression in CIF2-treated and untreated samples.
Fig. 4: SHR and CIF2 act at different stages of root development.
Fig. 5: SCR is not required for a response to CIF2.
Fig. 6: Ectopic SHR in the cortex and the endodermis with and without CIF2 treatment.

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Acknowledgements

We thank the Duke University Genome Sequencing and Analysis Core for sequencing the RNA libraries. We thank I. Efroni for assistance in the ICI pipeline and G. Wachsmann for assistance in mapping RNA-seq reads. We are also grateful to N. Clark and R. Sozzani for assistance with the paired correlation function technique. We thank the Duke Light Microscopy Facility and R. Ursache for assistance in microscopy, members of the Benfey lab for comments on the manuscript, V. G. Doblas for assistance with the CIF2 treatments, H. Belcher and J. Zhang for technical assistance and K. Gallagher for thoughtful discussion. This research was funded by grants from the NIH (R01-GM043778) and the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (GBMF3405) to P.N.B. N.G. was funded by an ERC Consolidator Grant (GA-no. 616228—ENDOFUN) and an SNSF grant (31003A_156261). Additional support was provided to C.D. through an EMBO short-term fellowship, to E.E.S. through startup funds from the University of Delaware, to P.M. by a FEBS long-term fellowship and to T.G.A. by an IEF Marie Curie fellowship.

Author information

Affiliations

Authors

Contributions

C.D., P.N.B. and N.G. designed the experiments. C.D. and P.N.B. wrote the manuscript. C.D. carried out the CIF2 treatments, barrier assays, FDA assays, staining/clearing, cell sorting and RNA-seq preparation. C.D., E.E.S. and I.T. carried out the RNA-seq analyses. C.D. and P.M. carried out the live-imaging experiments. C.D., T.G.A. and J.H.H. generated the transgenic lines. All authors contributed to the discussion and interpretation of results.

Corresponding author

Correspondence to Philip N. Benfey.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–15 and Supplementary Video legends.

Reporting Summary

Supplementary Table 1

Differentially expressed genes and CPMs of CIF2 treated FACS samples.

Supplementary Table 2

Index of Cell Identity results and statistics.

Supplementary Table 3

Differentially expressed genes and CPMs of CIF2 treated whole root samples.

Supplementary Table 4

Paired Correlation Function analysis and statistics.

Supplementary Table 5

Ectopic CASP1 intensity measurements.

Supplementary Table 6

Sequencing mapping statistics.

Supplementary Table 7

Cell counting and statistical analysis.

Supplementary Table 8

Sequences of cloning primers.

Supplementary Video 1

FDA penetration of pWER::SHR-GFP without CIF2. Sum projections of live imaging of FDA application to pWER::SHR-GFP roots without CIF2 treatment. Scale bars, 50 μm. Time after FDA addition is indicated at the top left.

Supplementary Video 2

FDA penetration of pWER::SHR-GFP with CIF2. Sum projections of live imaging of FDA application to pWER::SHR-GFP roots treated with CIF2. Scale bars, 50 μm. Time after FDA addition is indicated at the top left.

Supplementary Video 3

Dynamics of ectopic CASP1-mCherry in pWER::SHR-GFP over time. Maximum projection of CASP1-mCherry (gray channel). Scale bar, 50 μm, time elapsed indicated in bottom right.

Supplementary Video 4

Stability of ectopic CASP1-mCherry in pWER::SHR-GFP over after removing CIF2. Maximum projection of CASP1-mCherry (gray channel). Scale bar, 25 μm, time after removing CIF2 indicated in bottom right.

Supplementary Video 5

Effect of CIF2 on CASP1-mCherry in differentiated cells of pWER::SHR-GFP. Maximum projections of CASP1-mCherry (yellow channel) in differentiated cells of pWER::SHR-GFP. Scale bar, 50 μm, time after adding CIF2 indicated in bottom right.

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Drapek, C., Sparks, E.E., Marhavy, P. et al. Minimum requirements for changing and maintaining endodermis cell identity in the Arabidopsis root. Nature Plants 4, 586–595 (2018). https://doi.org/10.1038/s41477-018-0213-y

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