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A phase-separated nuclear GBPL circuit controls immunity in plants

Nature volume 594, pages 424–429 (2021)Cite this article

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Abstract

Liquid–liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membraneless organelles compartmentalize diverse cellular activities in eukaryotes1,2,3. Here we identify a superfamily of plant guanylate-binding protein (GBP)-like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana, two members of this family—GBPL1 and GBPL3—undergo phase-transition behaviour to control transcriptional responses as part of an allosteric switch that is triggered by exposure to biotic stress. GBPL1, a pseudo-GTPase, sequesters catalytically active GBPL3 under basal conditions but is displaced by GBPL3 LLPS when it enters the nucleus following immune cues to drive the formation of unique membraneless organelles termed GBPL defence-activated condensates (GDACs) that we visualized by in situ cryo-electron tomography. Within these mesoscale GDAC structures, native GBPL3 directly bound defence-gene promoters and recruited specific transcriptional coactivators of the Mediator complex and RNA polymerase II machinery to massively reprogram host gene expression for disease resistance. Together, our study identifies a GBPL circuit that reinforces the biological importance of phase-separated condensates, in this case, as indispensable players in plant defence.

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Fig. 1: IDR-containing GBPLs are essential for plant defence.
Fig. 2: Defence activation triggers a GBPL1–GBPL3 chromatin switch for immune condensates in the nucleus.
Fig. 3: Arabidopsis GBPL3 immune condensates are assembled via LLPS.
Fig. 4: GDACs concentrate RNAPII coactivators and nuclear GBPL3 on promoters of Arabidopsis defence genes.

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Data availability

The original raw RNA-seq data that support the findings of this study have been deposited and made publicly available in the NCBI Gene Expression Omnibus with accession number GSE134651. RNA-seq data are provided in Supplementary Tables 4 and 5. Full versions of all gels and blots are provided in Supplementary Fig. 1. The phylogenetic source data related to Extended Data Fig. 1b and high-resolution images of Fig. 4c are available on Dryad from https://doi.org/10.5061/dryad.g1jwstqqv. Source data are provided with this paper.

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Acknowledgements

We thank all members of the MacMicking laboratory for feedback and discussions; X. Liu for TEM assistance; members of the J. Liu laboratory (R. Park, M. Shao and Y. Chang) for instruction on CLEM and FIB milling; S. Wu for cryo-ET data collection advice; and X. Li, N. Clay, J. Parker, X. Dong, J. Zhou, Q. Wu, L. Wu, F. Xu and others for sharing materials and tools. The MacMicking laboratory is supported by grants from NIH NIAID (R01AI068041-12, R01AI108834-05). J.D.M. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, New Haven, CT, USA

    Shuai Huang, Shiwei Zhu, Pradeep Kumar & John D. MacMicking

  2. Yale Systems Biology Institute, West Haven, CT, USA

    Shuai Huang, Shiwei Zhu, Pradeep Kumar & John D. MacMicking

  3. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    Shuai Huang, Shiwei Zhu, Pradeep Kumar & John D. MacMicking

  4. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Shuai Huang, Shiwei Zhu, Pradeep Kumar & John D. MacMicking

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  1. Shuai Huang
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  2. Shiwei Zhu
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  4. John D. MacMicking
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Contributions

S.H. performed most experiments and analyses throughout with the following exceptions. P.K. purified human GBP1 and GBP5 proteins and undertook TLC assays. S.Z. performed negative-stain electron microscopy and cryo-ET (including FIB and CLEM). J.D.M. and S.H. conceived the project, designed experiments and wrote the manuscript with input from all authors.

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Correspondence to John D. MacMicking.

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Extended data figures and tables

Extended Data Fig. 1 Retrieval and structural characteristics of plant IDR-containing GBPLs.

a, Pipeline of IDP-based in silico screening of the Arabidopsis proteome. b, Phylogenetic tree inferred by Bayesian analysis. Support value: Left, Nodal posterior probabilities; Right, Maximum likelihood bootstrap values. Scale bar, substitutions per site. Number of amino acid (#aa) not drawn to scale. c, 3D structure prediction of Arabidopsis GBPLs performed on I-TASSER web server and projected with PyMOL software. GD, GTPase domain. HD, helical domain. IDR, intrinsically disordered region. hGBP1, 1F5N.PDB. d, Domain configuration and size of the Arabidopsis GBPL family versus human GBP1. e, GBPL disorder and hydrophobicity plots carried out using ProtScale and PONDR servers, respectively. Charged amino acids (red, glutamate/ aspartate; blue, arginine/lysine) displayed as vertical lines atop panels. IDRs are shaded.

Extended Data Fig. 2 Generation and characterization of Arabidopsis gbpl mutants.

a, Immunoblot of GBPL3 protein level using α-GBPL3 rabbit polyclonal antibody in wild type (WT) (Col-0) and four independent T1 transformants generated via CRISPR/Cas9 targeting GBPL3 (predicted molecular weight 122 kDa). Ponceau S staining of a non-specific band (nsb) serves as a loading control. Proteins extracted from five-week-old plants. Experiment performed once only due to the limit of tissue materials from sterile plants. b, PAM sequences of gRNAs and the consequences of CRISPR-Cas cleavages in T1 generation. Line 15 is biallelic and line 16 is heterozygous. c, Chromatograms of sanger sequencing of the mutation sites in CRISPR lines in b. d, Fresh weight of plants (mean ± SD) shown above (top-right graph the same as Fig. 1b for comparison with left). e, Gene expression of GBPL1 and GBPL2 in T-DNA mutants. f, Immunoblot (α-GBPL3) of GBPL3 and EGFP–GBPL3 levels in complemented plants. Ponceau S staining of RbcL serves as a loading control. g, Growth of Psm ES4326 (Psm) at day 3 of infection. Inoculum, bottom left. h, Hpa Noco2 sporulation in 2~3-week-old plants at day 7. Inoculum, bottom left. i, GBPL proteins are not required for Pst DC3000 Avr challenge using gbpl mutant lines. Growth of Pst-Avr at day 3 of infection. Inoculum, bottom left. g–i, Box = 25th and 75th percentiles; bars = min and max values. Statistical analysis, comparison of mean via one-way ANOVA test (Bonferroni post hoc correction). ns, not significant. Individual data points represent biologically independent samples (d, g–i).

Source data

Extended Data Fig. 3 RNA-seq and defence-gene expression analysis in transgenic plants.

a, Comparative heat map of the top 1,000 differentially expressed genes (RNA-seq; base log2) across Arabidopsis genotypes from two biologically independent replicates. b, GO enrichment of upregulated genes in Col-0/pGBPL3::GBPL3 (GBPL3OX) (left) and gbpl1-1 (right) plants versus WT. Top 25 significantly enriched GO terms are shown. Red, enriched GO-terms found in both GBPL3OX and gbpl1-1 plants. c, d, Defence-gene expression (mean ± SD) in under basal and induced conditions. RNA extracted 4 h after mock (MgCl2) or Psm ES4326 (OD600 = 0.1) infection. Expression normalized to ACTIN7. e, f, Defence-gene expression (mean ± SD) under basal and induced conditions shown as log2 transformations for comparison. g, h, Relative expression (mean ± SD, normalized to ACTIN7) in two different gbpl3 mutants. RNA extracted 24 h after mock (MgCl2) or Psm ES4326 (OD600 = 0.001) infection from 4-week-old plants. Statistical analysis, two-tailed Student’s t-test (c, e), one-way ANOVA with Holm–Sidak post hoc test (g) and two-way ANOVA with Holm–Sidak post hoc test (d, f, h). Individual data points represent biologically independent samples with experiments undertaken twice with similar results (c–h).

Source data

Extended Data Fig. 4 Nuclear GBPL1–GBPL3 analysis in Arabidopsis and N. benthamiana.

a, Live imaging of EGFP–GBPL3 in the nucleus of 2-week-old transgenic Col-0 or gbpl1-1 plants under basal conditions. Bar, 5 μm. b, Sequential fractionation strategy to examine chromatin binding properties of GBPL1 and GBPL3 in Fig. 2. using Col-0/pGBPL1:GBPL1-3×Flag plants to ensure physiological expression. c, Nuclear co-immunoprecipitation of GBPL3 with FLAG-M2 agarose (bottom) and GBPL1 with α-GBPL3 antibody (top). Streptavidin agarose and rabbit normal IgG served as negative controls. d, Split luciferase complementation assay in N. benthamiana. (top) Luminescence image of N. benthamiana leaves co-infiltrated with agrobacterial strains containing plasmids shown below. (bottom) Quantification of LUC activity (mean ± SD, n = 4 biologically independent leaf discs). e, Time course of total GBPL3 levels by immunoblot after salicylic acid treatment (0.5 mM). Ponceau S stained RbcL serves as loading control. f, Immunoblot of total GBPL3 protein levels 24 h after Psm ES4326 (OD600 = 0.01) infection from 4-week old plants. g, Top, Exclusion of mRFP-GBPL1 to the periphery of EGFP–GBPL3 nuclear condensates when co-expressed in N. benthamiana leaves. Scale bar, 2 μm. Inset bar used for average line profiling. Bottom, Average line profile (2 μm) of fluorescence intensity spanning GBPL3 condensates. Bar = mean ± s.d. (n = 5 biologically independent condensates). Shaded area with arrows, GBPL3 condensate core.

Source data

Extended Data Fig. 5 GBPL droplets are unique LLPS structures conserved across plant species, cell types and during development.

a, Coomassie staining of purified rEGFP protein. b, Standard curve of rEGFP via confocal microscopy. c, Quantitative measurement (mean ± SD) of EGFP–GBPL3 concentration 24 h after salicylic acid (0.5 mM), Psm (OD600 = 0.001) and Pst (OD600 = 0.001) from multiple 4-week-old plant nuclei. Comparison of mean via one-way ANOVA test with Holm–Sidak post hoc test. Individual data points represent biologically independent samples. d, e, Colocalization analysis of EGFP–GBPL3 in N. benthamiana (d, live cell) and HeLa cells (e, fixed cell). The latter enabled parallel antibody detection of subnuclear structures. DSB, double strand break. DNA stained with Hoechst 33342 dye. Bar, 5 μm (d, N.B.) and 2 μm (e, HeLa). f, Immunofluorescence of native GBPL3 in Col-0 and Col-0/GBPL3OX plants. Nuclei were isolated from 3-week-old soil-grown plants and DNA stained with Hoechst 33342. chr, chromocentre (white arrows). Bar, 2 μm. Experiment repeated once with similar results. g, Left, GBPL3 condensates in Arabidopsis cotyledons stably expressing 35S::EGFP-GBPL3. Shown are maximum projection images. Dashed circle, nuclear boundary. Bar, 5 μm. Right, salicylic acid-induced GBPL3 condensates (mean ± SD, Student’s t-test, two-tailed) in guard cells from 10-day-old Arabidopsis cotyledons (n = 4 biologically independent plants with 100 guard cells each). Mock, water treatment. h, Localization of EGFP-tagged tomato and maize GBPs in HeLa cells. DNA stained with Hoechst 33342 dye. Bar, 5 μm (SlGBP2 and ZmGBP1) and 2 μm (SlGBP1 and ZmGBP2). i, Maximum intensity projection images of EGFP–GBPL3 transiently expressed in N. benthamiana (N.B.), human HeLa and HEK 293T cells. Bar, 2 μm. j, Immunogold transmission electron microscopy of spheroid GDACs (dashed circles) expressed in HEK 293T cells. NE, nuclear envelope. NPC, nuclear pore complex. Bar, 500 nm. k, GBPL3 condensates were sensitive to 1,6-hexanediol (5%) but not 1,2,6-hexanetriol (5%). Digitonin (10 μg/ml) was used to facilitate chemical delivery.

Source data

Extended Data Fig. 6 Comparative LLPS behaviour of GBPLs in cells and cell-free systems.

a, A 3D volume view of rEGFP–GBPL3 droplets (10% Ficoll) depicted in (Fig. 3d). Bar, 2 μm. b, Phase separation (PS) diagram of rEGFP–GBPL3 across NaCl concentrations 30 min after reconstitution. c, Confocal images showing GBPL3 condensates fusion behaviours every 100 s in vitro. Images collected 30 min after reconstitution in droplet buffer (20 mM HEPES [pH  7.5], 200 mM NaCl, 1 mM TCEP, 10% Ficoll). Bar, 5 μm. d, RFP-GBPL1 does not generate LLPS nuclear condensates in situ in N. benthamiana. e, in vitro phase separation of rRFP–GBPL1 (375 nM) in droplet buffer (20 mM HEPES [pH  7.5], 150 mM NaCl, 1 mM TCEP). Coomassie brilliant blue (CBB) of rRFP–GBPL1 is shown on the left. Bar, 10 μm. f, Co-condensation assay of rEGFP–GBPL3 and rRFP–GBPL1 in vitro. Below, quantification of EGFP–GBPL3 droplet size. Median (solid line) and quartile (dashed line) values. Comparison of mean via one-way ANOVA with Holm–Sidak post hoc test (****P < 1.0 × 10−15, n = number of biologically independent droplets). g, Native PAGE of rEGFP−GBPL3 in the presence of increasing concentrations of rRFP−GBPL1 leads to GBPL3 displacement. Same loading controls below the original (anti-GFP) and re-probed (anti-RFP) native gel separated by SDS–PAGE. h, Exclusion of rRFP−GBPL1 from larger rEGFP−GBPL3 droplets in co-condensation assays. i, Representative negative stain EM images of rGBPL3 droplets at 24 h. Bar, 2 μm. Inset at right. Bar, 100 nm. j, FRAP analysis of 2 h old rEGFP−GBPL3 droplets in vitro. Bar, 5 μm. Fluorescence recovery of 5 individual droplets shown. Insert, representative images of a bleached droplet over 10 min recovery.

Source data

Extended Data Fig. 7 In situ cryo-ET analysis of GDACs including CLEM corroboration.

a, Flow chart of in situ cryo-ET analysis. b, c, CLEM images of EM grids seeded with HEK 293T cells transfected with CMV-EGFP-GBPL3. Single cell view of (b) shown below (c). These nuclear GDAC structures form in a species- and kingdom-independent manner with similar structures generated by GBPL3 plant orthologues; hence they appear to share conserved features with those directly generated in Plantae. d, Schema of FIB-milled lamella. e, CLEM images of FIB-milled lamella and overlay with Cryo-ET montage images. f, Left, Selected regions for tomograph collection. Right, Slice tomogram of GDACs and the corresponding CLEM fluorescence of GDACs overlap. g, h, Enlarged and higher resolution (binning factor 1) view of the GDAC in tomographic overlays with CLEM for region 1 of the cryo-ET montage image.

Extended Data Fig. 8 In situ cryo-ET analysis of GDACs in larger tomographic slices.

a, Shared similarities of CLEM-verified GDACs (from Extended Data Fig. 7h) with GDACs identified in tomographic images harbouring the nearby nuclear envelope for validating its intranuclear location (right, from Fig. 3e for comparison). Possible chromatin boundaries also depicted in both independently identified GDAC structures. b, CLEM images of EM grids seeded with HEK 293T cells transfected with CMV-EGFP-GBPL3 used to generate the tomogram in (a, right) above. Single cell view of the nucleus used to detect GDACs in far-right panel. Bar, 20 μm. c, FIB-milled lamella of the chosen nucleus from the CLEM above. d, Tomographic slice of the demarcated rectangular area 62 yielded a single demarcated GDAC near the nuclear envelope boundary shown in (a, right) and yielded 3D segmented images in Fig. 3e.

Extended Data Fig. 9 GBPL3 IDR contributes to LLPS-driven defence.

a, Top, domain structures of the Arabidopsis GBPL3 protein. Sites for mutagenesis are shown below. Bottom, GTP-binding pocket with conserved G-boxes (hGBP1 surface representation; 1F5N.PDB). GBPL3 hydrophobicity and disorder plots on right. Charged amino acids (red, glutamate and aspartate; blue, arginine and lysine) displayed as vertical lines atop panels. IDRs are shaded light green. b, Amino acid compositions of GBPL3 full length protein. Each row represents information for a single amino acid. Vertical bars, occurrence of indicated amino acid at that position. Right, amino acid percentage. c, Immunofluorescence of EGFP−GBPL3 mutant variants in HeLa cells 36 h after transfection. DNA stained with Hoechst 33342 dye. Arrows, nucleus. d, Growth of Psm ES4326 (Psm) and Pst DC3000 (Pst) at day 3 of infection. Inoculum, bottom left. Statistical analysis, comparison of mean via one-way ANOVA test (Bonferroni post hoc correction). ns, not significant. Box = 25th and 75th percentiles; bars = min and max values. Individual data points represent biologically independent samples. e, In vitro phase separation of rEGFP−GBPL3IDR and rEGFP. Equimolar (400 nM) amounts incubated at 22 °C for 16 h in droplet buffer (20 mM HEPES, pH  7.5, 150 mM NaCl, 1 mM TCEP, 10% Ficoll). Bar, 20 μm. Left, Coomassie stain. f, Immunofluorescence of plant−human GBP-IDR chimeras performed as in c.

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Extended Data Fig. 10 GBPL3 GTPase activity is required for LLPS-driven defence.

a, Alignment of conserved G-boxes in animal GBPs and plant GBPLs. Red outline denotes non-functional G2 box alterations in GBPL1. b, GTP hydrolysis assay of recombinant proteins along with tag controls. Bottom, percent GTP conversion. c, Malachite green phosphate assay (mean ± SD, n = 2 biologically independent samples) of recombinant GBPL proteins. d, Top, Immunoblot (α-GBPL3) of GBPL3 and EGFP–GBPL3K83A,S84A levels in Col-0 and Col-0/35S::EGFP-GBPL3K83A,S84A plants. T1-6 used in Extended Data Fig. 9d. Ponceau S staining of RbcL serves as a loading control. Bottom, Nuclear fractionation and immunoblot analysis of EGFP−GBPL3K83A,S84A in HEK 293T cells shows the DN GTPase mutant still enters the nucleus and binds chromatin but cannot phase separate. Sn, nuclear soluble. CB, chromatin bound. e, Live imaging of 35S::EGFP-GBPL3 and 35S::EGFP-GBPL3K83A,S84A in N. benthamiana and Arabidopsis plants. Arabidopsis imaging 24 h after salicylic acid (0.5 mM) treatment. Arrows, nucleolus. Bars, 5 μm (N.B.) and 2 μm (Arabidopsis). f, Effect of EGFP−GBPL3K83A,S84A (ks mt) on wild-type (WT) GBPL3 in confocal microscopy. Plasmids were co-transfected in HeLa cells and analysed 36 h post transfection. Bar, 2 μm. g, GBPL3 self-assembly co-IP in HEK 293T cells reveal GBPL3K83A,S84A can bind WT GBPL3 to interfere with its nuclear LLPS function in f. Cells were collected 36 h post transfection.

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Extended Data Fig. 11 GBPL3 enlists a monopartite NLS motif to target the nucleus where it engages Mediator subunits and excludes CDK8 for LLPS-driven immunity.

a, NLS motifs in GBPL3. Monopartite and bipartite NLS motifs were predicted via two web servers (SeqNLS and cNLS mapper). Shared sequences in red. b, Subcellular fractionation of gbpl3-3/35S::EGFP-GBPL3ΔNLS plants. RbcL (Ponceau staining) and Histone H3 are cytosol and nuclear controls, respectively. c, Live cell imaging of gbpl3-3/35S::EGFP-GBPL3∆NLS plant leaves under basal conditions. Image overlay of GFP and DIC channels. Arrows, cytosolic condensates formed by EGFP–GBPL3∆NLS. Bar, 10 μm. d, Immunofluorescence of CMV::EGFP-GBPL3ΔNLS in HeLa cells. Bar, 20 μm (top) and 5 μm (bottom). e, Spatiotemporal model derived from GBPL3 functional mutagenesis analysis. f, Combinatory GBPL3 interactome in Arabidopsis from co-IP candidates using Col-GBPL3-Flag plants under basal conditions and publicly available data sets (http://plants.proteincomplexes.org/). Node size denotes degree of protein–protein interactions. Classes of GBPL3 interactors grouped by colour-coding. Bracket denotes Mediator complex interactions. g, Different subunits associated with each region of the Arabidopsis Mediator complex (head [yellow], middle [green] and tail [blue] modules) and CDK8 kinase module. h, Line profile (colocalization) of fluorescence intensity for CDK8, MED15, MED19a and MED21 co-expressed with GBPL3 in (Fig. 4b). These head, middle and tail Mediator subunits directly overlap and interact with GBPL3 in leaf cell PPI profiling. CDK8 is excluded and surrounds GBPL3, as seen in line profiling. i, Effects of digitonin (10 μg/ml), digitonin plus 1,6-hexanediol (Hex, 5%), DRB (100 μM) and JQ1 (1 μM) on GBPL3 LLPS in live HeLa cells 2 h after treatment (n = 30 biologically independent cells/treatment). One-way ANOVA test with Bonferroni post hoc correction.

Source data

Supplementary information

Supplementary Figure 1

This file contains the gel source data.

Reporting Summary

Supplementary Table 1

NLS sequences of candidate nuclear IDPs (only one NLS is shown).

Supplementary Table 2

Gene family classification of candidate nuclear IDPs.

Supplementary Table 3

Domain accretion of plant GBPLs uncovered from NCBI.

Supplementary Table 4

Differentially expressed genes in GBPL3-OX plants compared with WT (padj-value < 0.05 and log2(Fold change) > 1.5).

Supplementary Table 5

Differentially expressed genes in gbpl1-1 plants compared with WT (padj-value < 0.05 and log2(Fold change) > 1.5).

Supplementary Table 6

Reported mediator activities in Arabidopsis immunity.

Supplementary Table 7

A list of primers used in this study.

Peer Review File

Video 1 : Live cell imaging of in situ formation of GBPL3 nuclear condensates (event 1).

Soil-grown gbpl3-3/35S::EGFP-GBPL3 plants were treated with 0.5 mM SA and imaging of in situ GBPL3 condensates formation in leaf nuclei was performed 5 h after treatment. Frame interval, 15 s. Bar, 2 μm.

Video 2 : Live cell imaging of in situ formation of GBPL3 nuclear condensates (event 2).

Soil-grown gbpl3-3/35S::EGFP-GBPL3 plants were treated with 0.5 mM SA and imaging of in situ GBPL3 condensates formation in leaf nuclei was performed 5 h after treatment. Frame interval, 10 s. Bar, 2 μm.

Video 3 : Live cell imaging of in situ formation of GBPL3 nuclear condensates (event 3, GFP channel).

Soil-grown gbpl3-3/35S::EGFP-GBPL3 plants were treated with 0.5 mM SA and imaging of in situ GBPL3 condensates formation in leaf guard cell nuclei was performed 5 h after treatment. Frame interval, 10 s. Bar, 2 μm.

Video 4 : Live cell imaging of in situ formation of GBPL3 nuclear condensates (event 3, merged channels).

Soil-grown gbpl3-3/35S::EGFP-GBPL3 plants were treated with 0.5 mM SA and imaging of in situ GBPL3 condensates formation in leaf guard cell nuclei was performed 5 h after treatment. Frame interval, 10 s. Bar, 2 μm.

Video 5 : Live cell imaging of fusion events of GDACs in Arabidopsis.

Confocal imaging (GFP channel) of GBPL3 condensates fusion (arrows) was performed in 10 d-old Arabidopsis cotyledon guard cells expressing EGFP-GBPL3. Frame interval, 5 s. Bar, 5 μm.

Video 6

: 3D tomographic reconstruction of cryo-ET analysis of GDACs. Scale bar, 200 nm.

Video 7 : 3D tomographic reconstruction and overlay of the segmented structures.

Ribosomes and vesicles are evident in the cytoplasm whereas GDAC is detected only in the nucleoplasm region. NE, Nuclear envelope. NPC, Nuclear pore complex.

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Huang, S., Zhu, S., Kumar, P. et al. A phase-separated nuclear GBPL circuit controls immunity in plants. Nature 594, 424–429 (2021). https://doi.org/10.1038/s41586-021-03572-6

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