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Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity

Nature Plants volume 8, pages 802–816 (2022)Cite this article

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Abstract

Plants use cell-surface immune receptors to recognize pathogen-specific patterns to evoke basal immunity. ENHANCED DISEASE SUSCEPTIBILITY (EDS1) is known to be crucial for plant basal immunity, whereas its activation mechanism by pattern recognition remains enigmatic. Here, we show that the fungal pattern chitin induced the plasma membrane-anchored receptor-like cytoplasmic kinase PBS1-LIKE 19 (PBL19) to undergo nuclear translocation in Arabidopsis. The palmitoylation-deficient PBL19C3A variant constantly resided in the nucleus, triggering transcriptional self-amplification mainly through WRKY8 and EDS1-dependent constitutive immunity. Unexpectedly, the metacaspase-cleaved PBL19 lacking the N-terminal nuclear localization sequence specifically interacted with and phosphorylated EDS1 in the cytoplasm. Phosphodeficient EDS1 attenuated PBL19C3A-induced constitutive immunity, while phosphomimetic EDS1 complemented the loss of PBL19 for fungal resistance. Collectively, these findings reveal a compelling model wherein the plasma membrane, nuclear and cytoplasmic pools of PBL19 temporally coordinate distinct roles of immune signal receiver, amplifier and effector to boost plant antifungal immunity via EDS1.

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Fig. 1: Chitin induces nuclear translocation of PBL19.
Fig. 2: PBL19C3A induces constitutive immunity and transcriptional self-amplification.
Fig. 3: WRKY8 is involved in transcriptional self-amplification of PBL19.
Fig. 4: EDS1 is a key player in PBL19-mediated immunity.
Fig. 5: Metacaspase MC4 may mediate ntPBL19 generation.
Fig. 6: Cytoplasmic accumulation of EDS1 is induced by chitin elicitation or phosphomimetic mutations.
Fig. 7: Working model for PBL19-mediated immunity in Arabidopsis.

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Data availability

The RNA-seq data reported in this paper can be found at the Gene Expression Omnibus (GEO): GSE188663. The ChIP–seq data reported in this paper can be accessed at https://figshare.com/articles/dataset/ChIP_seq_zip/14215772. The Arabidopsis TAIR10 genome database can be accessed at http://www.arabidopsis.org. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31970278 and 32125004) and the Fundamental Research Funds for the Central Universities in China (19lgzd32) to J.-F.L. and partially by the National Natural Science Foundation of China (31900305) to F.-Z.W and by the Sun Yat-sen University research grant (33000-31143406) to S.X. The authors are grateful to J. Sheen for critical comments on the manuscript, K. He for sid2-3 mutant seeds, H. Cui for eds1-2 mutant seeds and H. Guo and J. Huang for the V. dahliae strain V592.

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Author notes

  1. These authors contributed equally: Yujia Li, Jiao Xue.

Authors and Affiliations

  1. State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Yujia Li, Jiao Xue, Feng-Zhu Wang, Xiangjuan Huang, Ben-Qiang Gong, Yuheng Tao, Wenzhong Shen, Kehan Tao, Nan Yao, Shi Xiao & Jian-Feng Li

  2. Guangdong Provincial Key Laboratory for Crop Germplasm Resources Preservation and Utilization, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, China

    Jiao Xue

  3. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Jian-Min Zhou

  4. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China

    Jian-Min Zhou

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  1. Yujia Li
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Contributions

J.-F.L. conceived the study and designed the experiments. Y.L., J.X., F.-Z.W., X.H., B.-Q.G., Y.T. and W.S. performed experiments. Y.L., J.X., K.T., J.-F.L., J.-M.Z., S. X. and N.Y. analysed the data. J.-F.L. supervised the study and wrote the manuscript.

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Correspondence to Jian-Feng Li.

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Extended data

Extended Data Fig. 1 PBL19 possesses a functional nuclear localization sequence.

a, PBL19 contains a predicted palmitoylation site at Cys3 (blue) and a putative nuclear localization sequence (NLS) (red), whereas its paralogs PBL20 and BIK1 only have a palmitoylation site (blue). b, PBL20 and PBL20C3A are localized to the PM and cytoplasm, respectively, in protoplasts. HY5-Cherry is the nuclear marker. Scale bar = 10 μm. c, The NLS of PBL19 is functional. Note that the N-terminal fragment of PBL19 containing the NLS is sufficient to drive some cytosolic mCherry-GUS proteins into the nucleus. Dof1a-GFP is the nuclear marker. Scale bar = 10 μm. The experiments in (b) and (c) were repeated twice with similar results.

Extended Data Fig. 2 Chitin induces nuclear translocation of PBL19.

a, Chitin induces a fraction of PBL19 proteins to translocate from the PM to the nucleus. The same protoplasts transiently expressing PBL19-GFP were immobilized by the thin alginate layer and photographed before and after 1 h chitin exposure. The experiments were repeated twice with similar results. b,c, Biochemical fractionation indicates that nuclear translocation of PBL19 in transgenic plants only occurs under the elicitation of chitin (b) but not flg22 (c). d, Biochemical fractionation indicates that chitin does not elicit nuclear translocation of transiently expressed PBL20 in protoplasts.

Source data

Extended Data Fig. 3 The nuclear translocation of PBL19 is biologically relevant.

a, PBL19 and PBL20 function redundantly in chitin-induced MAPK activation. Four-week-old plants were treated with 500 μg ml-1 chitin for 15 min. Rubisco staining indicates equal protein loading. The experiments were repeated three times with similar results. b, pbl19 null plants are more susceptible than wild-type (WT) and pbl20 null plants to V. dahliae infection. c, pbl19 null plants and PBL19C3A,nls complementation plants exhibit comparable susceptibility to V. dahliae infection.

Extended Data Fig. 4 Nuclear-localized PBL19 induces transcriptional self-amplification and defence-related gene expression.

a, RNA-seq analysis reveals that defence-related genes are upregulated in pbl19/pPBL19:PBL19C3A-GFP-HA plants. b, RNA-seq analysis reveals that PBL19 (red) is the most highly upregulated gene in the RLCK VII subfamily in PBL19C3A complementation plants. c, RT–qPCR assay confirms the upregulation of defence marker genes in PBL19C3A complementation plants. ACTIN2 was used as the reference gene for normalizing indicated gene expression. Data are shown as mean ± s.d. of three biological replicates with P values indicated (two-sided Student’s t-test). d, RNA-seq and ChIP–seq co-analysis reveals that PBL19 (red) is upregulated by PBL19C3A and meanwhile is bound by the nuclear-localized PBL19. e, PBL19 transcription is upregulated by chitin treatment for 30 min. ACTIN1 was used as the reference gene for normalizing PBL19 expression. Data are shown as mean ± s.d. of three biological replicates with P values indicated (two-sided Student’s t-test).

Extended Data Fig. 5 WRKY8 and WRKY28 are transcriptionally upregulated by PBL19C3A.

a, Four closely related WRKYs in the WRKY IIc clade. b,c, RNA-seq (b) and RT–qPCR (c) assays reveal that these WRKYs are transcriptionally upregulated in PBL19C3A complementation plants. d, WRKY48 and WRKY71 are unable to increase the activity of the PBL19 promoter (pPBL19). e, WRKY8 and WRKY28 are unable to increase the activity of a mutated PBL19 promoter (pPBL19Δwbox) lacking all W-boxes. In (d) and (e), UBQ10-GUS was used in the protoplast-based promoter-LUC assay for normalizing transfection efficiency. The basal pPBL19 or pPBL19Δwbox activity was normalized as 1 in each replicate. In (c)–(e), data are shown as mean ± s.d. of three biological replicates with P values indicated (two-sided Student’s t-test).

Extended Data Fig. 6 PBL19C3A interacts with and phosphorylates WRKY8 and WRKY28.

a, BiFC assay reveals that PBL19C3A constitutively interacts with WRKY8 and WRKY28 in protoplasts. HY5-mCherry labels the nucleus. IAA28 was used to replace WRKYs as a negative control. Scale bar = 5 μm. b, In vitro kinase assay indicates that PBL19 can phosphorylate WRKY8 and WRKY28 but not WRKY48. The kinase-dead (Km) PBL19 is a negative control. c, PBL19-mediated WRKY8 phosphorylation sites identified by mass spectrometry. Identified phosphosites with peptide ion scores over 20 are considered reliable. Mass error in parts per million (ppm) = [1-(experimentally determined mass)/(predicted mass)]×106. d, Thr177 and Thr227 (red) are conserved residues in the DNA-binding domain of WRKY8 and WRKY28. Note that Thr209 and Thr210 (black) of WRKY8 can be phosphorylated by CPK5. e, Phosphomimetic T177D/T227D mutations can enhance the potency of WRKY8 in activating the PBL19 promoter. The basal pPBL19 activity was normalized as 1 in each replicate. Data are shown as mean ± s.d. of three biological replicates with P values indicated (one-way ANOVA with Tukey’s multiple comparisons test). The experiments in (a) and (b) were repeated three times with similar results.

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Extended Data Fig. 7 WRKY8 and WRKY28 positively regulate plant antifungal immunity.

a, CRISPR-mediated knockout of WRKY8 and WRKY28 generates multiple null alleles. The protein length of wild-type or mutated WRKY8 and WRKY28 is indicated in the parenthesis. b,c, wrky8-c3 wrky28-c2 double mutant plants are more susceptible to V. dahliae infection. Disease severity in each datapoint was quantified by qPCR-based quantification of fungal biomass in pooled above-ground tissues of 32 plants from indicated genotype. Data are presented as boxplots with each dot representing the datapoint of one biological replicate. For the boxplots, the central line indicates the median, the bounds of the box show the 25th and the 75th percentiles, and the whiskers indicate 1.5 × interquartile range between the 25th and the 75th percentiles. P value was indicated (two-sided Student’s t-test). d, BiFC assay reveals that WRKY28 interacts with WRKY8 in protoplasts. ARF5 was used to replace WRKY8 as a negative control. Scale bar = 10 μm.

Extended Data Fig. 8 ntPBL19 and PBL19Δ1-24 interact with EDS1 in cytoplasmic punctate structures.

a, BiFC assay reveals that EDS1 interacts with PBL19C3A in cytoplasmic punctate structures in protoplasts. The nucleocytoplasmic protein InLYP1 was used to replace PBL19C3A as a negative control. b, Coomassie brilliant blue (CBB) staining of IP-enriched ntPBL19 proteins from pbl19/pPBL19:PBL19C3A-GFP-HA plants. c, PBL19Δ1-24 is mainly localized to the cytoplasm in protoplasts. Scale bar = 10 μm. d, BiFC assay reveals that EDS1 interacts with PBL19Δ1-24 in cytoplasmic punctate structures in protoplasts. Scale bar = 10 μm. e, PBL19Δ1-24 proteins in multiple transgenic lines exhibit comparable molecular sizes to ntPBL19. Rubisco staining indicates comparable protein loading. f, PBL19Δ1-24 complementation plants are only slightly smaller than wild-type plants. Representative four-week-old plants are shown. Scale bar = 1 cm. g, CRISPR-mediated knockout of MC4~MC7 in PBL19C3A complementation plants. The experiments in (a), (c) and (d) were repeated three times and those in (b) and (e) were repeated twice with similar results.

Extended Data Fig. 9 Cytoplasmic accumulation of EDS1 is induced by treatment of chitin but not flg22.

a, The anti-EDS1 antibody can recognize endogenous EDS1 as a protein slightly bigger than 60 kDa. Rubisco staining indicates comparable protein loading. b. The anti-EDS1 antibody can recognize endogenous EDS1 that is co-immunoprecipitated with ntPBL19. c-e, Chitin induces cytoplasmic accumulation of endogenous EDS1 in a PBL19-dependent manner. Chitin-induced cytoplasmic enrichment of EDS1 only occurred in wild-type (c) and pbl19/pPBL19:PBL19-GFP-HA (e) plants but not in pbl19 null (d) plants. f, flg22 is unable to induce cytoplasmic accumulation of endogenous EDS1. In (c)–(f), the asterisks mark endogenous EDS1 in the blots. The abundances of EDS1 were quantified by Image J based on the densitometric ratios against tubulin (Input fraction), histone H3 (Nucleic-enriched fraction), and tubulin (Nucleic-depleted fraction), respectively. All experiments were repeated at least twice with similar results.

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Extended Data Fig. 10 Chitin-induced EDS1 phosphorylation by PBL19 promotes antifungal immunity.

a, PBL19-mediated EDS1 phosphorylation sites in vitro identified by mass spectrometry. Identified phosphosites with peptide ion scores over 20 are considered reliable. Mass error in parts per million (ppm) = [1-(experimentally determined mass)/(predicted mass)]×106. b, Phosphodeficient 5A mutations of EDS1 cannot fully abolish PBL19-mediated phosphorylation in vitro. GST was used as a control. c, Four in vitro phosphorylation sites (orange) are located on the protein surface of EDS1. Note that another phosphorylation site (Thr623) corresponds to the last residue of EDS1 and is located in a disordered tail missing in the deciphered structure. d,e, Phos-tag electrophoresis reveals that chitin-induced EDS1 phosphorylation depends on the kinase activity of PBL19 (d) and is dramatically reduced in phosphodeficient EDS15A (e). Indicated proteins were transiently expressed in pbl19 null protoplasts. f,g, Phosphomimetic EDS15D tends to be enriched in the cytoplasm relative to phosphodeficient EDS15A in pbl19 null plants (f) or protoplasts (g). In (f) and (g), the abundances of EDS1 or derivatives were quantified by Image J based on the densitometric ratios against tubulin (Input fraction), histone H3 (Nucleic-enriched fraction), and tubulin (Nucleic-depleted fraction), respectively. h, Phosphomimetic EDS15D can suppress the susceptibility of pbl19 null plants to V. dahliae infection. i, Complementary expression of EDS15D enhances plant resistance to V. dahliae relative to wild-type plants, whereas that of EDS15A impairs plant resistance. The experiments in (b) and (g) were repeated twice and those in (d), (e) and (f) were repeated three times with similar results.

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Li, Y., Xue, J., Wang, FZ. et al. Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity. Nat. Plants 8, 802–816 (2022). https://doi.org/10.1038/s41477-022-01195-x

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