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Plant HEM1 specifies a condensation domain to control immune gene translation

Nature Plants volume 9, pages 289–301 (2023)Cite this article

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Abstract

Translational reprogramming is a fundamental layer of immune regulation, but how such a global regulatory mechanism operates remains largely unknown. Here we perform a genetic screen and identify Arabidopsis HEM1 as a global translational regulator of plant immunity. The loss of HEM1 causes exaggerated cell death to restrict bacterial growth during effector-triggered immunity (ETI). By improving ribosome footprinting, we reveal that the hem1 mutant increases the translation efficiency of pro-death immune genes. We show that HEM1 contains a plant-specific low-complexity domain (LCD) absent from animal homologues. This LCD endows HEM1 with the capability of phase separation in vitro and in vivo. During ETI, HEM1 interacts and condensates with the translation machinery; this activity is promoted by the LCD. CRISPR removal of this LCD causes more ETI cell death. Our results suggest that HEM1 condensation constitutes a brake mechanism of immune activation by controlling the tissue health and disease resistance trade-off during ETI.

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Fig. 1: HEM1 is a translational regulator.
Fig. 2: hem1 promotes cell death to restrict pathogen growth during ETI.
Fig. 3: HEM1 inhibits the TE of pro-death immune genes.
Fig. 4: HEM1 controls translation through a plant-specific LCD.
Fig. 5: In vitro phase separation behaviours of HEM1.
Fig. 6: The LCD is required to inhibit ETI cell death.

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Data availability

All materials are available from the corresponding author upon request. All data generated or analysed during this study are included in this article and its Extended Data. The RNA-seq/Ribo-seq data generated in this study have been deposited in Gene Expression Omnibus under accession number PRJNA808663. Arabidopsis genome annotation version Ensemble V39 was used in this study. Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (no. 32070284), the Major Project of Hubei Hongshan Laboratory (no. 2022hszd016), the Key Research and Development Program of Hubei Province (no. 2022BFE003) and the start-up fund from Wuhan University to G.X. We thank X. Peng at Wuhan University for sharing the actin staining protocol. We thank X. Dong at Duke University for sharing the mutant information.

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Author notes

  1. These authors contributed equally: Yulu Zhou, Ruixia Niu, Zhijuan Tang.

Authors and Affiliations

  1. State Key Laboratory of Hybrid Rice, Institute for Advanced Studies (IAS), Wuhan University, Wuhan, China

    Yulu Zhou, Ruixia Niu, Zhijuan Tang, Rui Mou, Zhao Wang, Sitao Zhu & Guoyong Xu

  2. School of Life Sciences, Wuhan University, Wuhan, China

    Hongchun Yang

  3. Hubei Hongshan Laboratory, Wuhan, China

    Hongchun Yang & Guoyong Xu

  4. Institute of Biology Leiden, Leiden University, Leiden, the Netherlands

    Pingtao Ding

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  1. Yulu Zhou
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Contributions

Y.Z. and G.X. designed the experiment. R.N. performed all bioinformatics analyses. Z.T. performed the ribosome footprinting and polysome profiling experiments. Z.T. and R.M. optimized the ribosome footprinting protocol. R.M. screened the dst5 mutant and generated the CRISPR parental plasmid. Z.W. helped with the screening of the CRISPR editing lines. S.Z. and H.Y. helped with the LC–MS/MS analysis. P.D. helped with the data analysis. Y.Z. performed the rest of the experiments. G.X. and Y.Z. wrote the paper with input from all authors.

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Correspondence to Guoyong Xu.

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Extended data

Extended Data Fig. 1 HEM1 is the causal gene for the dst5 mutant phenotype.

a, Schematic of the uORFs-LUC reporter and representative pictures of the basal LUC activity. TBF1 exon1 with uORFs 1/2 and sequence of the N-terminal 73 amino acids was in-frame fused with firefly luciferase (LUC) driven by constitutive CaMV 35 S promoter. NOS ter, NOS terminator. Translational reporter uORFs-LUC/Col-0 was used for the EMS mutagenesis screen. dst5, a mutant with increased LUC activity from M3 generation. C1 and C2, two independent genetic complementation lines of dst5 with a genomic region of the HEM1 expression cassette. T-DNA, a SALK_135634 insertion line with a homozygous uORFs-LUC reporter. Scale bars, 1 cm. b, Whole-genome resequencing identified three closely-linked homozygous mutations on chromosome 2, Chr. 2: C11572722T (AT2G27100), Chr. 2: C14798110T (AT2G35110/HEM1), and Chr. 2: C15262299T (AT2G36380) in the dst5 mutant. Arrowheads indicate the gene orientation on the chromosome. Gene models, two gene models annotated on the TAIR website; red lines, introns; brown boxes, 5' leader and 3' UTR regions; green boxes, CDS regions with dashed boxes signifying the different CDSes in the two gene models. RNA-seq read coverage from TAIR JBrowser was further confirmed in our cDNA amplification assay and RNA-seq/Ribo-seq data. All evidence supported AT2G35110.2 as the dominant transcript model. The hem1 mutant is caused by a mutation on one splicing acceptor site, which leads to the retention of an intron and the gaining of an early stop codon in this retained intron. c, HEM1-related mutant information. d, HEM1’s acronyms and role in WAVE complex-mediated actin nucleation regulation. e, Profiling HEM1 expression pattern among different tissues by RT-qPCR. The top and bottom lines of the box plot represent the 25th and 75th percentiles, the center line is the median, and the whiskers are the full data range (n = 6). f, RNA-seq and Ribo-seq to show early translational termination on the hem1 mutant. No Ribo-seq read was found after the mutation site because of the premature stop in the retained intron indicated by the blue dashed rectangle.

Extended Data Fig. 2 HEM1 in immune responses.

a, MAPK activation. Three-week-old plants were infiltrated with 1 μM elf18 solution and collected at the indicated time points for immunoblot analysis using the phosphospecific antibody against MAPK3 and MAPK6. CBB, coomassie brilliant blue staining. b, Callose deposition. Three-week-old plants were infiltrated with 1 μM elf18 or Mock. Scale bars 100 μm. c, The elf18-induced resistance to Psm ES4326. Mean ± s.d. (n = 8). d, e, mYFP-HEM1 under its native promoter complements the LUC (d, upper; scale bars, 1 cm), growth (d, middle; scale bars, 1 cm) and trichome (d; bottom; scale bars, 1 mm), and ETI cell death (e; scale bars, 1 cm) phenotypes of hem1. efr-1, defective in the elf18 recognition receptor EFR. Line1/2, two independent genetic complementation lines with mYFP-HEM1 under its native promoter in the hem1 background.

Source data

Extended Data Fig. 3 Optimization of ribosome footprinting method.

a, LUC induction after ETI activation. WT and hem1 plants carry the isogenic Dex:AvrRpt2 cassette. Data are mean ± s.d. of the absolute LUC grey value post ETI induction by Dex treatment (n = 24). b, Polysome profiling of WT and hem1 without (Mock) or with ETI induction (Dex). c, Cutting preference of RNase T1 compared to RNase I. Nucleotides of 3' read ends were counted. Mean ± s.d. (n = 4). d, Mapping ratio of RF read to nuclear protein-encoding genes using RNase T1 in this study and RNase I (Hsu et al., 2017). Mean ± s.d. (n = 4). Two-sided Student’s t-test. e, Read distribution of RS (upper) and RF (bottom) around start (left) and stop codons (right) using 5' read ends. f, Correlation between two replicates (Rep1/2) of RNA-seq (RS) and Ribo-seq (RF) samples of WT and hem1. Data are shown as the correlation of log2RPKM in CDS for expressed genes with RPKM in CDS ≥ 1. r, Pearson correlation coefficient tested by two-sided Student’s t-test. g, Formulations of the transcriptome (RSfc), translatome (RFfc) and translation efficiency (TEfc) changes from the previous state (denominator) to the current state (numerator). Statistical methods used to describe the significance of each change are parenthesized.

Extended Data Fig. 4 Global ribosome footprinting analysis of hem1 without ETI activation.

a, TE of the LUC translational reporter. Ribo-seq coverage on the LUC CDS region was normalized to RNA-seq as 1 for WT and hem1. TE was increased by 19% in hem1. b, Transcriptome changes (RSfc) of hem1 compared to WT. Blue dots, transcriptionally downregulated genes (RSdn; for example, genes in immune response); Pink dots, transcriptionally upregulated genes (RSup; for example, genes in cytoskeleton). P value, Wald test. c, d, GO analysis of RSup (c) and RSdn (d) genes; e, Translation efficiency changes (TEfc) of hem1 compared to WT. Blue dots, translationally downregulated genes (TEdn; for example, genes in immune response); Pink dots, translationally upregulated genes (TEup; for example, genes in cell cycle). f, g, GO analysis of TEup (c) and TEdn (d) genes. P value (c, d, f, g), Hyper Geometric test.

Extended Data Fig. 5 Global ribosome footprinting analysis of hem1 in ETI responses.

a, Heatmap to show RSfc and RFfc of Zones 2/4 genes in WT and hem1 upon ETI induction. Numbers in the heatmap, genes found in WT-only, hem1-only, or shared in WT and hem1; color scale: log2 fold changes. b, GO analysis of WT-only, hem1-only, or shared Zone 4 genes in (a). c, GO analysis of genes with RFdn in hem1 but RFnc in WT upon ETI induction. d, e, GO analysis of HEM1-positively- (HEM1(+); d) or negatively- (HEM1(−); e) regulated genes through TE grouped according to RFfc in WT. P value (b–e), Hyper Geometric test.

Extended Data Fig. 6 Characterization of HEM1 and its protein interactome.

a, Representative silver staining to show HEM1 interactome. 35 S:GFP/Col-0, GFP driven by CaMV 35 S constitutive promoter in Col-0 background. ProHEM1:mYFP-HEM1/hem1, the genetic complementation line with mYFP-HEM1 driven by its native promoter in hem1 mutant. Immunoprecipitation (IP) with GFP-trap against GFP tag was performed to identify the HEM1 protein interactome whose components were detected at least in two out of three biological replicates while not detected in two replicates of two GFP control. b, GO analysis of the biological processes of HEM1 protein interactors using the online tool ShinyGO v0.741. Two clusters of translation and actin-related processes were detected. c, Phylogenetic tree to show the plant (w/ LCD; right half with a pink background) and animal (w/o LCD; left half with a blue background) family of HEM proteins. Animal HEM proteins could be further divided into HEM1/2 subfamilies (for example, Human), while plants have one (for example, Arabidopsis) or duplicated (for example, wheat) HEM1 proteins. d, Prediction of LCD with seven representative HEM1 homologs of plants (upper) and animals (bottom) using IUPred2 (https://iupred2a.elte.hu/) indicated by Prion domain (PrD)-like score. Domain organizations were searched against the PFAM database. e, The conservation of LCDs among plant HEM1s. Blue rectangle, the conserved regions with red color highlighting the consistent residues. f, 3D protein structures of AtHEM1 (left; AT2G35110.1) and HsHEM2 (right; NM_013436.5) stored in Alphafold2 database. The LCD region’s start, middle, and end positions are marked by arrowheads with corresponding positions of AT2G35110.2-encoded HEM1 in the parentheses.

Source data

Extended Data Fig. 7 HEM1 condensation behaviors in plants during immune activation.

a, ETI induction in N. benthamiana by recognition between β-estradiol-controlled bacterial effector AvrPto and the corresponding constitutively-expressed host target Pto. mYFP-HEM1 condensation by ETI activation through treatment with β-estradiol (+) with water as a control (–). Scale bars, 10 µm. b, mYFP-HEM1 condensation in N. benthamiana by SA at indicated concentrations and durations. Scale bars, 10 µm. c, Condensation behaviors of different mYFP-tagged HEM1 regions. Data are shown as mean ± s.d. (n = 10) of condensate numbers in each construct. One-way ANOVA with post-hoc analysis by Tukey test. d, HEM1ΔLCD and LCD alone decrease the condensation capacity by SA, and LCD alone has mislocalization to an unknown ring structure (arrowhead). Assays were performed in N. benthamiana. Scale bars, 10 µm. e, f, Detecting HEM1 condensates in the pellet fraction after SA treatment by microscopy (e) and immunoblot analysis of HEM1 (f). Scale bars, 5 µm. Experiments have been repeated three times with similar results. g, h, GO term of HEM1 interactome identified in the soluble (g) and pellet (h) fractions after SA treatment using the online ShinyGO v0.741. P value (g, h), Hyper Geometric test. g, Venn diagram of the HEM1 interactome in the soluble and pellet fractions. Translation factors are shown.

Source data

Extended Data Fig. 8 HEM1 condensation with translation factors during immune activation by SA treatment.

a, Interaction of HEM1 with nCBP and eIF3d-2 by BiFC. Transient expression of nYFP-HEM1 with nCBP-cYFP or eIF3d-2-cYFP was done in N.benthamiana. Scale bars, 10 µm. b, Co-localization assay to show LCD-dependence for co-existence in the condensate. Transient expression of mYFP-HEM1 with nCBP-CFP or eIF3d-2-CFP was done in N.benthamiana. Arrowheads indicate condensates. Scale bars, 10 µm. c, nCBP condensation in Arabidopsis by SA treatment depends on HEM1. 35 S:YFP-nCBP transgenic plants were generated and crossed into hem1. Scale bars, 10 µm. d, Immunoblot analysis of YFP-nCBP in the soluble and pellet fractions of Col-0 and hem1 after SA treatment. Experiments have been repeated three times with similar results.

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Extended Data Fig. 9 Gene editing by CRISPR.

a, Development of CRISPR-walking to identify the critical region responsible for HEM1-mediated translational control without interfering with actin function. The anchoring gRNA was paired with each gRNA walking in the same direction (upstream in the HEM1 study). b, Indel regions of independent ΔLCD, ΔC, Δhem1 and arp2 CRISPR editing lines. c, Representative pictures to show the growth (upper, scale bars, 1 cm) and trichome (bottom, scale bar = 1 mm) phenotypes of different mutants. d, Effects of inhibiting actin function by LAT-B on the LUC activity of the translational reporter. WT translational reporter plants were infiltrated with 5 μM LAT-B or Mock control. Leaves were stained with Phalloidin-iFluor-488 and examined for actin polymerization (upper; scale bars, 10 µm) under microscopy. Representative leaves show the LUC activity (bottom; scale bars, 1 cm) at the same time as the actin assay. e, A time-course recording of the LUC activity after LAT-B treatment in the WT leaves. Mean ± s.d. (n = 24).

Supplementary information

Supplementary Information

Supplementary Methods.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Ribosome footprinting analysis between WT and hem1. Supplementary Table 2: Ribosome footprinting analysis between WT and hem1 after ETI activation. Supplementary Table 3: HEM1 protein interactors. Supplementary Table 4: MS analysis of HEM1 interactors in the soluble and pellet fractions. Supplementary Table 5: Resource table.

HEM1 condensates.

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Source Data Extended Data Fig. 2

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Zhou, Y., Niu, R., Tang, Z. et al. Plant HEM1 specifies a condensation domain to control immune gene translation. Nat. Plants 9, 289–301 (2023). https://doi.org/10.1038/s41477-023-01355-7

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