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Pathogen-induced condensation of the guard cell RNA-binding protein SAIR1 fine-tunes translation for immunity

Nature Plants volume 11, pages 2548–2564 (2025)Cite this article

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Abstract

Plant guard cells perceive pathogens and close stomata to prevent their invasion. Biomolecular condensates are membraneless organelles essential for life processes. However, guard cell biomolecular condensates mediating stomatal immunity remain unknown. Here we identify a guard-cell-preferential RNA-recognition-motif-type RNA-BINDING PROTEIN, STOMATAL IMMUNE RNA-BINDING PROTEIN 1 (SAIR1), that forms pathogen-responsive guard cell condensates via phase separation. Upon perception of the pathogen molecular pattern flg22, the activated kinases MPK3 and MPK6 phosphorylate SAIR1 and trigger its condensation in guard cells for stomatal immunity. SAIR1 condensates recruit translational regulators such as POLYADENYLATE-BINDING PROTEINs and eIFiso4G, and sequester defence-related mRNAs, including key components of the salicylic acid pathway. Through these interactions, SAIR1 condensates enhance the translation of defence mRNAs, ultimately promoting stomatal closure. Our findings reveal phosphorylation-regulated SAIR1 condensates as a critical hub that links flg22–MPK3/6 signalling to stomatal immunity.

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Fig. 1: SAIR1 mediates stomatal immunity in Arabidopsis.
Fig. 2: SAIR1 undergoes phase separation and condensation.
Fig. 3: SAIR1 condensation is required for stomatal immunity.
Fig. 4: Phosphorylation of SAIR1 promotes its condensation for stomatal immunity during PTI.
Fig. 5: SAIR1 binds to defence-related mRNA in condensates.
Fig. 6: SAIR1 interacts with translation regulators and modulates defence-related translation.
Fig. 7: Phylogenetic analysis and schematic model of SAIR1.

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Data availability

Materials are available from the corresponding author upon request. The omics data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE286004. Source data are provided with this paper. All other data are provided in the main figures and the extended data figures.

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Acknowledgements

We thank J. Xu (Zhejiang University) for the MPK6SR seeds, X. Meng (Shanghai Normal University) for the Est::MKK5DD-HA seeds, W. Wang (Peking University) for helpful suggestions, and J. Ma and Z. Liu for technical assistance, and we thank the Protein Chemistry and Proteomics Facility and Center for Biomedical Analysis at Tsinghua University for technical support. We also thank the National Key Research and Development Program of China (grant no. 2024YFA0917100), the National Natural Science Foundation of China (grant no. 32370756 to T.Q.), the Project of State Key Laboratory of Tropical Crop Breeding (grant no. SKLTCBLHZD202501 to Wei Wang and T.Q.), the China Postdoctoral Science Foundation (grant no. 2025M772821 to Q.Y.), and the Tsinghua University Dushi Program (to T.Q.) for funding support.

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Authors and Affiliations

  1. State Key Laboratory of Green Biomanufacturing, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Qiangsheng Yu, Tianxue Song, Yuejuan Zeng & Tiancong Qi

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Jie Wu, Yunfan Jin, Haiteng Deng, Zhi John Lu & Xiaofeng Fang

  3. College of Life Sciences, Capital Normal University, Beijing, China

    Wenrui Wang & Susheng Song

  4. Plant Science and Technology College, Beijing University of Agriculture, Beijing, China

    Huang Huang

  5. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China

    Wei Wang & Jianghui Xie

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Contributions

T.Q., Q.Y., S.S. and X.F. conceived and conceptualized the study and designed the experiments. Q.Y., J.W., T.S. and Wenrui Wang performed the experiments. T.Q., Q.Y., H.H., Y.J., Z.J.L., H.D., Y.Z., Wei Wang and J.X. analysed the data. T.Q., Q.Y. and S.S. wrote the manuscript. All authors contributed to discussion and manuscript preparation.

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Correspondence to Susheng Song or Tiancong Qi.

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T.Q. and Q.Y. are listed as inventors on a patent application (CN202511361663.0) filed by Tsinghua University and based on this work.

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Nature Plants thanks Justin Lee, Maeli Melotto and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Screening for flg22-responsive proteins with intrinsically disordered regions (IDRs).

a, Workflow for screening IDR-containing proteins responsive to flg22 using b-isox. b, Enrichment of SAIR1 and its homolog SAIR2 by b-isox, showing increased b-isox/input ratios upon flg22 treatment.

Extended Data Fig. 2 Mutants, overexpression plants, and expression of SAIR1.

a, Schematics of SAIR1 and SAIR2. T-DNA insertion sites are indicated by triangles. b, GUS staining of abaxial leaf epidermal cells of 4-week-old pSAIR2::GUS plants. Scale bar, 20 μm. c, Phenotypes of the indicated genotypes. SAIR1 overexpression causes autoimmune dwarfism. Scale bar, 1 cm. d, Relative expression of SAIR1 in 4-week-old Col-0, sair1-1, and SAIR1 overexpression plants (SAIR1-YFP-OX1 and OX2) (n = 3). e, Relative expression of SAIR2 in 4-week-old Col-0 and sair2 plants (n = 3). f, Relative expression of SAIR1 in 4-week-old Col-0 and sair1-2 plants (n = 3). g, Stomatal apertures upon treatment with mock, Pst DC3000, or flg22 for 1 h. Sample sizes are indicated. h, Pathogen entry assays showing luminescent bacteria in leaves after 1 h exposure to Psm ES4326-lux. i, Bacterial growth determined at 3 d post-spray inoculation with Pst DC3000 (n = 8). j,k, Relative expression of SAIR1 in 4-week-old Col-0 plants under mock or Pst DC3000 treatment for 24 h (j), or under mock or flg22 treatment for 1 h (k) (n = 3). l, Bacterial growth determined at 3 d post-infiltration inoculation with Pst DC3000 (OD600nm = 0.0001) (n = 8). Individual data points are presented with means ± s.d. (d-g, j, k). n = independent biological replicates (d-f, i-l). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point (i, l). Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (d, g and l) or two-tailed t-test (e,f, i-k). The exact P values are provided in the Source Data file (d, g, l). The experiments were repeated three times with similar results (b, c, g, h).

Source data

Extended Data Fig. 3 Predictions of disordered regions in SAIR1 and SAIR2.

a, Disordered domain predictions by PONDR and PLAAC. IDRs and PrLDs are colored. b, Predicted structures of SAIR1 and SAIR2 from AlphaFold2.

Extended Data Fig. 4 SAIR1 interacts and functions together with SAIR2.

a, Bacterial growth determined at 3 d post-spray inoculation with Pst DC3000 (n = 8). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point.b, Phenotypes of 4-week-old indicated genotypes. Scale bar, 1 cm. c, Representative confocal images of guard cells from pSAIR1::SAIR1-YFP-1/sair1-1 at 1 h post-spray inoculation with mock or Pst DC3000. Scale bar, 2 μm. d, Representative confocal images of guard cells from SAIR2-YFP-OX2 in Col-0 or fls2 background at 1 h post-spray inoculation with mock or Pst DC3000. Scale bar, 5 μm. e, SAIR1 specifically forms condensates in guard cells. Leaves of SAIR1-YFP-OX1 were used. Scale bar, 50 μm (left panel), and 5 μm (right panel). f, Representative confocal images of guard cells from SAIR2-YFP-OX2 at 30 min post-infiltration with mock, nlp20, elf18 or chitin. Scale bar, 5 μm. g, Stomatal apertures upon treatment with mock, nlp20, elf18, or chitin for 1 h. Sample sizes are indicated. h, Phenotypes of 4-week-old Col-0 and SAIR2 overexpression plants (SAIR2-YFP-OX1 and -OX2). SAIR2 overexpression causes mild autoimmune phenotypes. Scale bar, 1 cm. i, Representative confocal images of guard cells from SAIR2-YFP-OX1 at 1 h post-spray inoculation with mock or Pst DC3000. Scale bar, 5 μm. j, Co-immunoprecipitation (Co-IP) assays showing the SAIR1-SAIR1 and SAIR1-SAIR2 interactions. IB, immunoblot. k, Yeast two-hybrid assays showing the SAIR1-SAIR1 and SAIR1-SAIR2 interactions. Individual data points are presented with means ± s.d. (g). n = independent biological replicates (a). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (a, g). The exact P values are provided in the Source Data file (a, g). The experiments were repeated three times with similar results (b-k).

Source data

Extended Data Fig. 5 Condensation of SAIR1 is required for stomatal immunity.

a, PrLD is essential for Pst DC3000-induced SAIR2 condensation. Scale bar, 20 μm.b, Protein expression of SAIR1-YFP or its variants in the indicated Arabidopsis transgenic lines. Ponceau S staining served as a loading control. c, Images of stomatal apertures of the indicated genotypes upon treatment with mock or Pst DC3000 for 1 h. Red lines show profiles of guard cells. Scale bar, 50 μm. d, Representative confocal images of guard cells from pSAIR1::SAIR1ΔIDR-FUS-YFP-1/sair1-1 at 1 h post-spray inoculation with mock or Pst DC3000. Scale bar, 5 μm. e, Phenotypes of 4-week-old plants of the indicated genotypes. Scale bar, 1 cm. f, Stomatal apertures at 1 h post treatment with mock or Pst DC3000. Sample sizes are indicated. g, Pathogen entry assays showing luminescent bacteria in leaves after 1 h exposure to Psm ES4326-lux. h, Bacterial growth determined at 3 d post-spray inoculation with Pst DC3000 (n = 8). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point.Individual data points are presented with means ± s.d. (f). n = independent biological replicates (h). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (f, h). The exact P values are provided in the Source Data file (f, h). The experiments were repeated three times with similar results (a-f, g).

Source data

Extended Data Fig. 6 SAIR1 phosphorylation is required for condensation and stomatal immunity.

a, SAIR1 protein sequence. Phosphorylated residues are shown in red. b, Co-IP assays showing the MPK3/6-SAIR1 interaction. 6×HA-tagged BAK1, MPK3, MPK6, MPK4 or MKK5 was co-expressed with SAIR1-YFP in Nb leaves. c, In vitro Phos-tag assays showing that the SAIR13A mutant impaired its phosphorylation by MPK3/6. The E. coli-expressed proteins were mixed as indicated. d, In vivo condensation of YFP-fused SAIR1, SAIR13A, and SAIR13D in Nb leaves at 0.5 h after infiltration with mock or flg22. Scale bar, 20 μm. e, Phenotypes of 4-week-old plants of the indicated genotypes. Scale bar, 1 cm. f, Stomatal apertures after 1 h treatment with mock or flg22. Sample sizes are indicated. g, Luminescent bacteria in leaves after 1 h exposure to Psm ES4326-lux. h, Bacterial growth determined at 3 d post-spray inoculation with Pst DC3000 (n = 8). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point.i, Representative confocal images of Nb epidermal cells expressing SAIR1-YFP with EV (Empty vector) or MKK5DD. Scale bar, 20 μm. j, Representative confocal images of guard cells in the indicated genotypes after 24 h treatment with mock or 5 μM β-estradiol. MKK5DD, a β-estradiol inducible MKK5DD expression line. Scale bar, 5 μm. k, l, Intermaps (k) and phase diagrams (l) of SAIR1 IDR and IDR3D predicted by FINCHES. m, Co-IP assays showing self-interactions of SAIR1, SAIR13A, and SAIR13D. Individual data points are presented with means ± s.d. (f). n = independent biological replicates (h). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (f, h). The exact P values are provided in the Source Data file (f, h). The experiments were repeated three times with similar results (b-g, i, j, m).

Source data

Extended Data Fig. 7 SAIR1 does not play critical roles in transcription and mRNA stability.

a, Volcano plots of differentially expressed genes in 3-week-old plants of the indicated genotypes at 24 h post-flood inoculation with Pst DC3000. FC, fold change.b, Heat map of relative expression of defense-related genes. c, Relative expression of defense-related genes under basal condition (-) or after 24 h flood-inoculation with Pst DC3000 (OD600nm = 0.2) (n = 3). d, SAIR1 minimally affects mRNA stability. 2-week-old seedlings were flood-treated with mock or 150 μg/mL cordycepin (polyadenylation inhibitor) for 1.5 h (n = 3). Individual data points are presented with means ± s.d. (c). n = independent biological replicates (c, d). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (c, d). The exact P values are provided in the Source Data file (c, d).

Source data

Extended Data Fig. 8 Global ribosome sequencing analysis in sair1-1 and Col-0 upon PTI activation.

a, Phenotypes of 4-week-old plants of the indicated genotypes. Scale bar, 1 cm. b, Bacterial growth determined at 3 d post-spray inoculation of Pst DC3000 (n = 8). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point.c, Stomatal apertures at 1 h post treatment with mock or Pst DC3000. Sample sizes are indicated. d, Representative confocal images of guard cells from indicated genotypes at 1 h post-spray inoculation with mock or Pst DC3000. Scale bar, 5 μm. e, Schematic of ribosome sequencing experimental design. f, Volcano plots of differential expressed genes under basal condition (-) or flg22-induced condition. g,h, GO analysis of mRNAs with increased ribosome-binding in ribosome profiling of Col-0 (g) or sair1-1 (h) upon flg22 treatment. i, GO analysis of mRNAs with decreased ribosome-binding in sair1-1 vs Col-0 under mock condition. Individual data points are presented with means ± s.d. (c). n = independent biological replicates (b). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (b, c). The exact P values are provided in the Source Data file (b, c). The experiments were repeated three times with similar results (a, c, d).

Source data

Extended Data Fig. 9 SAIR1-mediated stomatal immunity depends on SA pathway.

a, In vitro co-localization of SAIR1 condensates with 3’UTRs of AZI1, PEN3 and MPK11. CY5-labeled 3’UTRs were added to 10 μM SAIR1-GFP with 50 mM NaCl. Scale bar, 5 μm. b, Electrophoretic mobility shift assays (EMSAs) showing SAIR1 binding to 3’UTRs of AZI1, MPK11 and PEN3, with mannose binding protein (MBP) as a negative control. c, Dual-LUC assays in Arabidopsis protoplasts showing SAIR1-mediated translation of MPK11 and AZI1. LUC activities were measured after 45 min of mock (H2O) or 1 μM flg22 treatment on Col-0 or sair1-1 protoplasts with overnight expression of the indicated effectors and reporter (n = 3). BREm, mutated BRE. d, Bacterial growth in the indicated genotypes determined at 3 d post-spray inoculation with Pst DC3000 (n = 8). The box plots show the median (centre), the first and third quartiles (bounds of the box), and the whiskers extend to the furthest data point.e, The sid2, eds1, and pad4 mutations do not affect flg22-induced SAIR1 condensation in SAIR1-YFP-OX2 at 0.5 h post-infiltration with mock or flg22. f, Stomatal apertures upon treatment with Pst DC3000 for 1 h. Sample sizes are indicated. Individual data points are presented with means ± s.d. (c,f). n = independent biological replicates (c,d). Different letters indicate significant differences by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05) (c,d,f). The exact P values are provided in the Source Data file (c, d, f). The experiments were repeated three times with similar results (a, b, e, f).

Source data

Extended Data Fig. 10 Conservation of SAIR1 and homologs among plants.

a, Alignment of SAIR1 homologs from Arabidopsis, Nb, G. max, O. sativa and S. lycopersicums. b, Structures of SAIR1 homologs predicted by AlphaFold2. c, IDR prediction for SAIR1 homologs by PONDR. d, Representative confocal images of Nb epidermal cells expressing SlSAIR1-GFP or SlSAIR1ΔIDR-GFP at 10 h after infiltration with mock or Pst DC3000. Scale bar, 10 μm. e, Relative expression of SlSAIR1 in 6-week-old S. lycopersicum TRV2-GUS and TRV2-SlSAIR1 (n = 4 independent biological replicates). Individual data points are presented with means ± s.d. Data were analyzed by two-tailed t-test. f, Phenotypes of S. lycopersicum TRV2-GUS and TRV2-SlSAIR1 at 3 d post-spray-treatment with Pst DC3000. Scale bar, 1 cm. The experiments were repeated three times with similar results (d, f).

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

The SAIR1-enriched RNAs according to RIP-seq.

Supplementary Table 2

The SAIR1-associated proteins according to IP–MS.

Supplementary Table 3

Ribo-seq and RNA-seq analysis for sair1 and Col-0 under mock or flg22 treatment.

Supplementary Table 4

Primers and probes used in this study.

Supplementary Video 1

Dynamics of SAIR1 condensates in tobacco.

Supplementary Video 2

Fusion of SAIR1 condensates in Arabidopsis.

Supplementary Video 3

Fission of SAIR1 condensates in Arabidopsis.

Source data

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Yu, Q., Wu, J., Jin, Y. et al. Pathogen-induced condensation of the guard cell RNA-binding protein SAIR1 fine-tunes translation for immunity. Nat. Plants 11, 2548–2564 (2025). https://doi.org/10.1038/s41477-025-02154-y

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