Abstract
SERRATE (SE) plays an important role in many biological processes and under biotic stress resistance. However, little about the control of SE has been clarified. Here we present a method named native chromatin-associated proteome affinity by CRISPR–dCas9 (CASPA–dCas9) to holistically capture native regulators of the SE locus. Several key regulatory factors including PHYTOCHROME RAPIDLY REGULATED 2 (PAR2), WRKY DNA-binding protein 19 (WRKY19) and the MYB-family protein MYB27 of SE are identified. MYB27 recruits the long non-coding RNA–PRC2 (SEAIR–PRC2) complex for H3K27me3 deposition on exon 1 of SE and subsequently represses SE expression, while PAR2–MYB27 interaction inhibits both the binding of MYB27 on the SE promoter and the recruitment of SEAIR–PRC2 by MYB27. The interaction between PAR2 and MYB27 fine-tunes the SE expression level at different developmental stages. In addition, PAR2 and WRKY19 synergistically promote SE expression for pathogen resistance. Collectively, our results demonstrate an efficient method to capture key regulators of target genes and uncover the precise regulatory mechanism for SE.
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Data availability
The ChIP–seq data involved in this study are available at the NCBI BioProject https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA874567 (ref. 56) and NCBI SRA https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA972317 (ref. 57). Detailed information on the CASPA–dCas9 pull-down assays is available in Supplementary Data 1 and the Supplementary Methods. Detailed information on the ChIP–seq data and the identified differentially enriched peaks and differential degree genes can be found in Supplementary Data 3. The accessible link for TAIR 10 is https://www.arabidopsis.org/. Source data are provided with this paper.
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Acknowledgements
We acknowledge the Protein and Proteomics Centre in the Department of Biological Sciences, National University of Singapore, for collecting the liquid chromatography with tandem mass spectrometry data. This work was supported by a grant from the Singapore Millennium Foundation (no. R154-000-639-592) to Y.A.Y. and by the Fundamental Research Funds for the Central Universities (grant nos 020814380167 and 020814380180) to B.S.
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W.C. and B.S. conceptualized the project. W.C., A.H., Y.C. and X.W. devised the methodology. W.C., T.Z. and D.C. managed the software. W.C., J.W., Y. Zheng and Z.W. conducted the investigation. W.C., D.L., G.W., W.Y. and Y. Zhao provided the resources. W.C. wrote the original draft of the paper. B.S. reviewed and edited the paper. Y.A.Y. and B.S. acquired the funding. Y.A.Y. and B.S. administered and supervised the project.
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Extended data
Extended Data Fig. 1 Capture of regulatory factors of SE.
a, Representative phenotypes of the CRISPR/dCas9-gRNA1, -gRNA2, -gRNA3 and -gRNA4 lines compared with wildtype (WT). Scale bar, 1 cm. b, The dCas9-6Myc enrichment at gRNA3 targeting locus. The y axis shows the calibrated relative ratio of bound DNAs to input DNAs after IP, and IgG serves as the negative control. The dCas9-6Myc enrichment at a random region of AGO1 promoter was used as the reference. Values are the means ± s.d. of three biological replicates. c, The enrichment of precipitated peptides in the CRISPR/dCas9-gRNA3 line compared with the non-gRNA control from LC‒MS/MS data by volcano plot. The x axis represents the peptide number change (peptide number in the non-gRNA control minus peptide number in the CRISPR/dCas9-gRNA3) and the y axis represents the normalized P value. Peptides specific in the CRISPR/dCas9-gRNA3 line (Specific-gRNA3) are shown in cyan and peptides specific in the non-gRNA control (Specific-control) are shown in red (p < 0.05 by one-way ANOVA). The p values for PAR2, WRKY19 and MYB27 peptides are 0.004, 0.020 and 0.038, respectively. d, Mass spectrometry analysis of PAR2, WRKY19 and MYB27 peptides. e-g, Gene structures (upper panel) and expressions (lower panel) of PAR2 (e), WRKY19 (f) and MYB27 (g) in par2-1, wrky19-2 (w19-2) and myb27-1 mutants, respectively. The Inverted triangles and blue triangles represent the relative position of the T-DNA and the position of the primers used for qRT-PCR analysis, respectively. #1, #2 and #3 in (e-g) represent different individual plants in the same T-DNA insertion lines. Values are the means ± s.d. of three biological replicates. In b and e-g: **p < 0.01, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4.
Extended Data Fig. 2 Representative phenotypes of different mutants and analysis of miRNAs formation in par2-1 and myb27-1 mutants.
a, Representative phenotypes of the par2-1 mutant as compared to WT. Photographs were taken of 3-wk-old seedlings. Scale bar, 1 cm. b, The representative leaves of par2-1, wrky19-2 and myb27-1 mutants as compared to WT and se-1. Photographs were taken for the second, third, and fourth true leaves of 3-week-old seedlings. Scale bar, 1 cm. c, Numbers of serrations per side of each leaf (n = 75) of par2-1, wrky19-2 and myb27-1 mutants as compared to WT and se-1. Values are the means ± s.d. (n = 75). d, Accumulation of miRNAs (miR157, miR162 and miR164) in the WT and par2-1 mutant by northern blotting. U6 snRNA was used as the control. e, qRT‒PCR analysis of target genes of the miRNAs in (d) in the WT and par2-1 mutant. Values are the means ± s.d. of three biological replicates. f, qRT‒PCR analysis of miRNAs (miR157, miR162 and miR164) in the WT and myb27-1 mutant. Values are the means ± s.d. of three biological replicates. g, qRT‒PCR analysis of target genes of the miRNAs in (f) in the WT and myb27-1 mutant. #1, #2 and #3 in (e-g) represent different individual plants in the same T-DNA insertion lines. Values are the means ± s.d. of three biological replicates. h, The comparison of SEAIR expressions between pSEAIR:SEAIR and pSEAIR:SEAIR plus p35S:PAR2 in N. benthamiana leaves. Values are the means ± s.d. of three biological replicates. i, The comparison of SEAIR expressions between pSEAIR:SEAIR and pSEAIR:SEAIR plus p35S:MYB27 in N. benthamiana leaves. #1 and #2 in (h-i) represent different individual plants from the same expressing group. Values are the means ± s.d. of three biological replicates. In c and e-i: *p < 0.05; **p < 0.01; ns: no significance, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4.
Extended Data Fig. 3 Representative phenotypes of par2-1 pPAR2:PAR2-6Myc and myb27-1 pMYB27:MYB27-GFP plants.
a, Representative phenotypes of par2-1 pPAR2:PAR2-6Myc (upper) and myb27-1 pMYB27:MYB27-GFP plants (lower). Scale bar, 1 cm. b-c, qRT-PCR analysis of SE expressions in par2-1 pPAR2:PAR2-6Myc (b) and myb27-1 pMYB27:MYB27-GFP plants (c). Values are the means ± s.d. of three biological replicates. d-e, Accumulation of miRNAs (miR157, miR162 and miR164) in par2-1 pPAR2:PAR2-6Myc (d) and myb27-1 pMYB27:MYB27-GFP plants (e). Values are the means ± s.d. of three biological replicates. f-g, qRT‒PCR analysis of target genes of the miRNAs in (d-e) in par2-1 pPAR2:PAR2-6Myc (f) and myb27-1 pMYB27:MYB27-GFP plants (g). #1, #2 and #3 in (b-g) represent different individual plants in the same transgenic lines. Values are the means ± s.d. of three biological replicates. In b-g: *p < 0.05; **p < 0.01; ns: no significance, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4.
Extended Data Fig. 4 Representative phenotypes of PAR2 OE and MYB27 OE lines.
a, Representative phenotypes of the PAR2 OE lines as compared to WT. The white arrows indicate the dark-green PAR2 OE plants. Scale bar, 1 cm. b, Representative dark-green phenotype of the PAR2 OE plant. Scale bar, 2 mm. c, Representative phenotypes of the Weak, Middle and Strong lines as compared to WT. Scale bar, 1 cm. d-h, Representative flower phenotypes of the Weak (d, e) and Middle (g, h) lines as compared to WT (f). Scale bar in (d, e and h), 1 mm and Scale bar in (f, g), 2 mm. i, Percentage of different phenotypes of PAR2 OE lines (n = 22). j-k, qRT-PCR analysis of PAR2 (j) and SE (k) expressions in rosette leaves of Weak, Middle and Strong lines compared with those in WT. (j) and (k) share the same legend. Values are the means ± s.d. of three biological replicates. l, The protein amounts of PAR2 in PAR2-GFP-6Myc OE (Weak, Middle and Strong) lines. WT and anti-H3 were used as the negative control and loading control, respectively. m, Representative phenotypes of the 27-Weak and 27-ox lines as compared to WT. The yellow and white arrows indicate the 27-Weak and 27-ox plants, respectively. Scale bar, 1 cm. n, Representative phenotypes of detached leaves of the WT and 27-ox lines. o, Percentage of different phenotypes of MYB27 OE lines (n = 54). p, qRT-PCR analysis of SE expressions in rosette leaves of 27-ox lines compared with those in WT. #1, #2 and #3 represent different individual plants in the 27-ox line. Values are the means ± s.d. of three biological replicates. In j, k and p: *p < 0.05; **p < 0.01, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4. The experiments in l were repeated independently at least twice with similar results.
Extended Data Fig. 5 The expression patterns of SEAIR, PAR2, MYB27, WRKY19 and PAR1 compared with SE during plant development and under biotic stresses.
a-f, The dynamic transcript level of SE (a), SEAIR (b), PAR2 (c), MYB27 (d), WRKY19 (e) and PAR1 (f) in rosette leaves along Arabidopsis growth time. Values are the means ± s.d. of three biological replicates. g-m, The dynamic transcript level of PR1 (g), SE (h), SEAIR (i), PAR2 (j), MYB27 (k), WRKY19 (l) and PAR1 (m) in rosette leaves along elf18 treatments. Values are the means ± s.d. of three biological replicates. n-t, The dynamic transcript level of PR1 (n), SE (o), SEAIR (p), PAR2 (q), MYB27 (r), WRKY19 (s) and PAR1 (t) in rosette leaves along flg22 treatments. Values are the means ± s.d. of three biological replicates.
Extended Data Fig. 6 The binding assays of WRKY19 to the SE promoter, SE mRNA signals by in situ hybridization assays in leaf cells, yeast two-hybrid assays between MYB27 and different components of PRC1/2 and the overlap of peaks with increased H3K27me3 in MYB27 OE and reduced H3K27me3 in myb27-1.
a, EMSA analysis between MBP-WRKY19 and the putative WRKY19-binding fragment (named pSE frag-1) in SE promoter. The full length of WRKY19-binding motif is shown in red. b, Yeast one-hybrid assay between WRKY19 and the 2000 bp SE promoter (pSE). The pSE without the W-box was named pSE mut. Transformed yeast cells were grown on media lacking tryptophan and histidine (SD/-Trp/-His). pHisi.1-0 or pDEST22-0 refers to empty only. 2 mM 3AT (3-Amino-1,2,4-triazole) was used for self-activation repression. c, SE mRNA signals by in situ hybridization assays in the WT and myb27-1 mutant. The red arrows indicated SE mRNA signals in leaf cells. The sense probe of SE was used as the negative control. Scale bar, 20 μm. d, Comparison of SE protein between pSE:SE-GFP and myb27-1 pSE:SE-GFP plants. Anti-H3 was used for the loading control. e, Yeast two-hybrid assays between MYB27 and different components of PcG complex. Transformed yeast cells were grown on media lacking leucine and tryptophan (SD/-Trp/-Leu) and lacking leucine, tryptophan, histidine, and adenine (SD/-Trp/-Leu/-His/-Ade). pGADT7-0 or pGBDK7-0 refers to empty only. f, Overlap of peaks with increased H3K27me3 in MYB27 OE and reduced H3K27me3 in myb27-1. The experiments in a and c-d were repeated independently at least twice with similar results.
Extended Data Fig. 7 The “SEAIR-PRC2” complex is recruited by MYB27 for SE repression.
a, Representative phenotypes of SEAIRa OE and myb27-1 SEAIRa OE plants as compared to WT. The red arrows indicate serrated leaves. Photographs were taken of 3-wk-old seedlings. Scale bar in (upper panel), 2 cm and Scale bar in (lower panel), 1 cm. b, The representative leaves of SEAIRa OE and myb27-1 SEIARa OE plants as compared to WT. Photographs were taken for the second, third, and fourth true leaves of 3-week-old seedlings. Scale bar, 1 cm. c, Numbers of serrations per side of each leaf of SEAIRa OE and myb27-1 SEAIRa OE plants as compared to WT. Values are the means ± s.d. (n = 75). In boxplots, centre lines and box edges are medians and the interquartile range (IQR), respectively. Whiskers extend within 1.5 times the IQR and the plus signs represent the means. d, H3K27me3 enrichment on SE chromatin in rosette leaves of 18-d-old seair-1, myb27-1 SEAIRa OE and SEAIRa OE plants as compared to WT by ChIP-seq assays. e, Changes in the enrichment of H3K27me3 on exon 1 of SE (P3) in rosette leaves of 18-d-old seair-1, myb27-1 SEAIRa OE and SEAIRa OE plants as compared to WT by ChIP-qPCR analyses. The P3 position of SE is shown in Fig. 3d. Values are the means ± s.d. of three biological replicates. f, qRT-PCR analysis of SE expressions in rosette leaves of seair-1, myb27-1 SEAIRa OE and SEAIRa OE plants as compared to WT. Values are the means ± s.d. of three biological replicates. g, Accumulation of miRNAs (miR157, miR162 and miR164) in rosette leaves of seair-1, myb27-1 SEAIRa OE and SEAIRa OE plants as compared to WT. Values are the means ± s.d. of three biological replicates. h, qRT‒PCR analysis of target genes of the miRNAs in (g) in rosette leaves of seair-1, myb27-1 SEAIRa OE and SEAIRa OE plants as compared to WT. Values are the means ± s.d. of three biological replicates. i, qRT-PCR analysis of SE expressions in rosette leaves of seair-1, myb27-1, myb27-1 seair-1 plants and WT. #1 and #2 represent different individual plants in the same lines. Values are the means ± s.d. of three biological replicates. j, RIP-qPCR analysis of 5’ truncation and 3’ truncation of SEAIR by using anti-GFP beads for pull-down in leaves from myb27-1 pMYB27:MYB27-GFP. The binding of MYB27-GFP on U6 was taken for the negative control and the relative enrichment rate was set to 1. Values are the means ± s.d. of three biological replicates. k, ChIRP assay of 5’ truncation of SEAIR on SE loci in rosette leaves of 18-d-old WT, myb27-1 and MYB27 OE (27-ox) with odd or even probe pools. Probes that target LacZ mRNA are served as the negative control. Odd probe pools: mix of probes 1 and 3 from 5’ truncation of SEAIR; even probe pools: mix of probes 2 and 4 from 5’ truncation of SEAIR. Positions of P2 and P4 of SE locus are shown in Fig. 3d. Values are the means ± s.d. of three biological replicates. l, H3K27me3 enrichment on SE chromatin in rosette leaves of 18-d-old SEAIRa OE and MYB27 OE plants. In c and e-k: *p < 0.05; **p < 0.01, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4.
Extended Data Fig. 8 The interaction between GST-PAR2 and MBP-MYB27 and the disruption of MYB27–FIE interaction by PAR2.
a, In vitro pull-down assays between GST-PAR2 and MBP-MYB27. The pull-down between GST and MBP-MYB27 was used as the negative control. b, CoIP assays between GFP/MYB27-GFP and PAR2-6Myc in Arabidopsis protoplasts. The proteins were precipitated by anti-GFP beads and detected by anti-GFP or anti-Myc antibody. c, Yeast one-hybrid assay between PAR2 and the 2000 bp SE promoter. Transformed yeast cells were grown on media lacking tryptophan and histidine (SD/-Trp/-His). pHisi.1-0 or pDEST22-0 refers to empty only. 2 mM 3AT was used for self-activation repression. Yeast one-hybrid assay between MYB27 and SE promoter was used as the positive control. d, The comparison of dual-luciferase assays between pSE:LUC plus p35S::GFP and pSE:LUC plus p35S:PAR2 in N. benthamiana leaves (upper panel). The relative intensity of luciferase was quantitively presented by qRT-PCR (lower panel). Values are the means ± s.d. of three biological replicates. e, The binding of PAR2-6Myc at SE promoter (P2) in rosette leaves of par2-1 pPAR2:PAR2-6Myc and par2-1 myb27-1 pPAR2:PAR2-6Myc plants. The y axis shows the calibrated relative ratio of bound DNAs to input DNAs after IP, and IgG serves as the negative control. #1 and #2 represent different individual plants in the same lines. Values are the means ± s.d. of three biological replicates. f, The disruption of MYB27–FIE interaction by PAR2 through yeast-three-hybrid assays. g, The disruption of MYB27–FIE interaction by PAR2 through BiLC assays. In d and e: **p < 0.01; ns: no significance, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4. The experiments in a and b were repeated independently at least twice with similar results.
Extended Data Fig. 9 The modulations of PAR2 and MYB27 fine-tune SE expressions during Arabidopsis development.
a-b, qRT-PCR analysis of PAR2 (a) and MYB27 (b) expressions in rosette leaves of 18-d-old and 30-d-old Arabidopsis. Values are the means ± s.d. of three biological replicates. c, Changes in the enrichment of H3K27me3 on exon 1 of SE (P3) in rosette leaves of 18-d-old and 30-d-old par2-1 mutants compared with WT. The y axis shows the calibrated relative ratio of bound DNAs to input DNAs after IP, and IgG serves as the negative control. The P3 position of SE is shown in Fig. 3d. Values are the means ± s.d. of three biological replicates. d, The enrichment of H3K27me3 on SE loci searching in H3K27me3 CHIP-seq database (http://www.plantseq.org/). e, Changes in the enrichment of H3K27me3 on exon 1 of SE (P3) in siliques, cauline leaves and 30-d-old rosette leaves of WT. The y axis shows the calibrated relative ratio of bound DNAs to input DNAs after IP, and IgG serves as the negative control. The P3 position of SE is shown in Fig. 3d. Values are the means ± s.d. of three biological replicates. f, qRT-PCR analysis of SE expressions in siliques, cauline leaves and 30-d-old rosette leaves of WT. (e) and (f) share the same legends. Values are the means ± s.d. of three biological replicates. g, The representative leaves of cauline leaves and 30-d-old rosette leaves. Scale bar, 1 cm. h-i, qRT-PCR analysis of PAR2 (h) and MYB27 (i) expressions in siliques, cauline leaves and 30-d-old rosette leaves of WT. (h) and (i) share the same legends. Values are the means ± s.d. of three biological replicates. In a-c, e-f and h-i: *p < 0.05; **p < 0.01, two-sided Student’s t-test. The exact p values are shown in Supplementary Data 4.
Supplementary information
Supplementary Information
Supplementary Table 1 and Methods.
Supplementary Data 1
Supplementary Data 1–4.
Source data
Source Data Fig. 1
Statistical source data for Fig. 1.
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Statistical source data for Fig. 2.
Source Data Fig. 3
Statistical source data for Fig. 3.
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Statistical source data for Fig. 4.
Source Data Figs. 4 and 5 and Extended Data Figs. 2, 4, 6 and 8
Unprocessed western blots and gels.
Source Data Fig. 5
Statistical source data for Fig. 5.
Source Data Extended Data Figs. 1–5 and 7–9
Statistical source data for Extended Data Figs. 1–5 and 7–9.
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Chen, W., Wang, J., Wang, Z. et al. Capture of regulatory factors via CRISPR–dCas9 for mechanistic analysis of fine-tuned SERRATE expression in Arabidopsis. Nat. Plants 10, 86–99 (2024). https://doi.org/10.1038/s41477-023-01575-x
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DOI: https://doi.org/10.1038/s41477-023-01575-x
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