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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.

References

  1. Zhou, J. M. & Zhang, Y. Plant immunity: danger perception and signaling. Cell 181, 978–989 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Boutrot, F. & Zipfel, C. Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance. Annu. Rev. Phytopathol. 55, 257–286 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Yu, X., Feng, B., He, P. & Shan, L. From chaos to harmony: responses and signaling upon microbial pattern recognition. Annu. Rev. Phytopathol. 55, 109–137 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jubic, L. M., Saile, S., Furzer, O. J., Kasmi, F. E. & Dangl, J. L. Help wanted: helper NLRs and plant immune responses. Curr. Opin. Plant Biol. 50, 82–94 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Lolle, S., Stevens, D. & Coaker, G. Plant NLR-triggered immunity: from receptor activation to downstream signaling. Curr. Opin. Immunol. 62, 99–105 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ngou, B. P. M., Ahn, H. K., Ding, P. & Jones, J. D. G. Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592, 110–115 (2021).

    CAS  PubMed  Google Scholar 

  7. Yuan, M. et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 592, 105–109 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186–194 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Bressendorff, S. et al. An innate immunity pathway in the moss Physcomitrella patens. Plant Cell 28, 1328–1342 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gong, B. Q., Wang, F. Z. & Li, J. F. Hide-and-seek: chitin-triggered plant immunity and fungal counterstrategies. Trends Plant Sci. 25, 805–816 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Cao, Y. et al. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife 3, e03766 (2014).

    Article  PubMed Central  Google Scholar 

  12. Liu, J. et al. A tyrosine phosphorylation cycle regulates fungal activation of a plant receptor Ser/Thr kinase. Cell Host Microbe 23, 241–253 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Gong, B. Q. et al. Cross-microbial protection via priming a conserved immune co-receptor through juxtamembrane phosphorylation in plants. Cell Host Microbe 26, 810–822 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Lu, D. et al. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl Acad. Sci. USA 107, 496–501 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, J. et al. Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7, 290–301 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Shinya, T. et al. Selective regulation of the chitin-induced defense response by the Arabidopsis receptor-like cytoplasmic kinase PBL27. Plant J. 79, 56–66 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Rao, S. et al. Roles of receptor-like cytoplasmic kinase VII members in pattern-triggered immune signaling. Plant Physiol. 177, 1679–1690 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bi, G. et al. Receptor-like cytoplasmic kinases directly link diverse pattern recognition receptors to the activation of mitogen-activated protein kinase cascades in Arabidopsis. Plant Cell 30, 1543–1561 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kadota, Y. et al. Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol. Cell 54, 43–55 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Li, L. et al. The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15, 329–338 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Tian, W. et al. A calmodulin-gated calcium channel links pathogen patterns to plant immunity. Nature 572, 131–135 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Thor, K. et al. The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity. Nature 585, 569–573 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yamada, K. et al. The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation. EMBO J. 35, 2468–2483 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu, Y. et al. Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure. eLife 8, e44474 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Tena, G., Boudsocq, M. & Sheen, J. Protein kinase signaling networks in plant innate immunity. Curr. Opin. Plant Biol. 14, 519–529 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, B., Meng, X., Shan, L. & He, P. Transcriptional regulation of pattern-triggered immunity in plants. Cell Host Microbe 19, 641–650 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chinchilla, D. et al. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–550 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Gao, M. et al. Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe 6, 34–44 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Pietraszewska-Bogiel, A. et al. Interaction of Medicago truncatula lysin motif receptor-like kinases, NFP and LYK3, produced in Nicotiana benthamiana induces defence-like responses. PLoS ONE 8, e65055 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Domínguez-Ferreras, A. et al. An overdose of the Arabidopsis coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or its ectodomain causes autoimmunity in a SUPPRESSOR OF BIR1-1-dependent manner. Plant Physiol. 168, 1106–1121 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Lapin, D., Bhandari, D. D. & Parker, J. E. Origins and immunity networking functions of EDS1 family proteins. Annu. Rev. Phytopathol. 58, 253–276 (2020).

    Article  CAS  PubMed  Google Scholar 

  32. Aarts, N. et al. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proc. Natl Acad. Sci. USA 95, 10306–10311 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Feys, B. J., Moisan, L. J., Newman, M. A. & Parker, J. E. Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J. 20, 5400–5411 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bartsch, M. et al. Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell 18, 1038–1051 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wirthmueller, L., Zhang, Y., Jones, J. D. G. & Parker, J. E. Nuclear accumulation of the Arabidopsis immune receptor RPS4 is necessary for triggering EDS1-dependent defense. Curr. Biol. 17, 2023–2029 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Cui, H. et al. A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity. New Phytol. 213, 1802–1817 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. García, A. V. et al. Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete plant innate immune response. PLoS Pathog. 6, e1000970 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Heidrich, K. et al. Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses. Science 334, 1401–1404 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Wiermer, M., Feys, B. J. & Parker, J. E. Plant immunity: the EDS1 regulatory node. Curr. Opin. Plant Biol. 8, 383–389 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Chen, G. et al. TaEDS1 genes positively regulate resistance to powdery mildew in wheat. Plant Mol. Biol. 96, 607–625 (2018).

    Article  CAS  PubMed  Google Scholar 

  41. Lipka, V. et al. Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis. Science 310, 1180–1183 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Fradin, E. F. et al. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol. 150, 320–332 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Moreau, M. et al. EDS1 contributes to nonhost resistance of Arabidopsis thaliana against Erwinia amylovora. Mol. Plant Microbe Interact. 25, 421–430 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Makandar, R. et al. The combined action of ENHANCED DISEASE SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, and SENESCENCE-ASSOCIATED101 promotes salicylic acid-mediated defenses to limit Fusarium graminearum infection in Arabidopsis thaliana. Mol. Plant Microbe Interact. 28, 943–953 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Wu, Y. et al. Loss of the common immune coreceptor BAK1 leads to NLR-dependent cell death. Proc. Natl Acad. Sci. USA 117, 27044–27053 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Park, C. J. & Ronald, P. C. Cleavage and nuclear localization of the rice XA21 immune receptor. Nat. Commun. 3, 920 (2012).

    Article  PubMed  CAS  Google Scholar 

  47. Lal, N. K. et al. The receptor-like cytoplasmic kinase BIK1 localizes to the nucleus and regulates defense hormone expression during plant innate immunity. Cell Host Microbe 23, 485–497 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hemsley, P. A. The importance of lipid modified proteins in plants. New Phytol. 205, 476–489 (2015).

    Article  CAS  PubMed  Google Scholar 

  49. Grebenok, R. J. et al. Green-fluorescent protein fusions for efficient characterization of nuclear targeting. Plant J. 11, 573–586 (1997).

    Article  CAS  PubMed  Google Scholar 

  50. Gao, F. et al. Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens. Nat. Plants 5, 1167–1176 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gao, X. et al. Bifurcation of Arabidopsis NLR immune signaling via Ca2+-dependent protein kinases. PLoS Pathog. 9, e1003127 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nawrath, C. & Métraux, J. P. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11, 1393–1404 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Shen, W., Liu, J. & Li, J. F. Type-II metacaspases mediate the processing of plant elicitor peptides in Arabidopsis. Mol. Plant 12, 1524–1533 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Chakraborty, J., Ghosh, P., Sen, S. & Das, S. Epigenetic and transcriptional control of chickpea WRKY40 promoter activity under Fusarium stress and its heterologous expression in Arabidopsis leads to enhanced resistance against bacterial pathogen. Plant Sci. 276, 250–267 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Bhattacharjee, S., Halane, M. K., Kim, S. H. & Gassmann, W. Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators. Science 334, 1405–1408 (2011).

    Article  CAS  PubMed  Google Scholar 

  57. Wagner, S. et al. Structural basis for signaling by exclusive EDS1 heteromeric complexes with SAG101 or PAD4 in plant innate immunity. Cell Host Microbe 14, 619–630 (2013).

    Article  CAS  PubMed  Google Scholar 

  58. Wong, J. E. M. M. et al. A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation. Proc. Natl Acad. Sci. USA 116, 14339–14348 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Chen, L., Zhang, L. & Yu, D. Wounding-induced WRKY8 is involved in basal defense in Arabidopsis. Mol. Plant Microbe Interact. 23, 558–565 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Wu, L. T. et al. Arabidopsis WRKY28 transcription factor is required for resistance to necrotrophic pathogen Botrytis cinerea. Afr. J. Microbiol. Res. 5, 5481–5488 (2011).

    Article  CAS  Google Scholar 

  61. Liebrand, T. W. H. et al. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc. Natl Acad. Sci. USA 110, 10010–10015 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Pruitt, R. N. et al. The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity. Nature 598, 495–499 (2021).

    Article  CAS  PubMed  Google Scholar 

  63. Jia, X., Zeng, H., Wang, W., Zhang, F. & Yin, H. Chitosan oligosaccharide induces resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana by activating both salicylic acid- and jasmonic acid-mediated pathways. Mol. Plant Microbe Interact. 31, 1271–1279 (2018).

    Article  CAS  PubMed  Google Scholar 

  64. Tateda, C. et al. Salicylic acid regulates Arabidopsis microbial pattern receptor kinase levels and signaling. Plant Cell 26, 4171–4187 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Tian, H. et al. Activation of TIR signaling boosts pattern-triggered immunity. Nature 598, 500–503 (2021).

    Article  CAS  PubMed  Google Scholar 

  66. Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  PubMed  Google Scholar 

  67. Xie, X. et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol. Plant 10, 1246–1249 (2017).

    Article  CAS  PubMed  Google Scholar 

  68. Pantelides, I. S., Tjamos, S. E. & Paplomatas, E. J. Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae. Mol. Plant Pathol. 11, 191–202 (2010).

    Article  CAS  PubMed  Google Scholar 

  69. Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nat. Plants 3, 930–936 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Gookin, T. E. & Assmann, S. M. Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors. Plant J. 80, 553–567 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Lei, R., Qiao, W., Hu, F., Jiang, H. & Zhu, S. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability. MethodsX 2, 24–32 (2015).

    Article  PubMed  Google Scholar 

  72. Kim, D. et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  74. Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Wu, J., Hettenhausen, C., Meldau, S. & Baldwin, I. T. Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. Plant Cell 19, 1096–1122 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequencing data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Zhang, Y. et al. Model-based analysis of ChIP–Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Kaufmann, K. et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP–SEQ) or hybridization to whole genome arrays (ChIP–CHIP). Nat. Protoc. 5, 457–472 (2010).

    Article  CAS  PubMed  Google Scholar 

  80. Yang, Q. et al. The receptor-like cytoplasmic kinase CDG1 negatively regulates Arabidopsis pattern-triggered immunity and is involved in AvrRpm1-induced RIN4 phosphorylation. Plant Cell 33, 1341–1360 (2021).

    Article  PubMed  Google Scholar 

  81. Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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