Calcium-sensing receptor AcCaS regulates chloroplast immunity in kiwifruit by competitively binding with Ca²⁺ or the Psa effector

Abstract

Activating chloroplast immunity to enhance host resistance offers a novel and sustainable approach for the effective control of kiwifruit bacterial canker. Chloroplasts serve as a central hub for ROS, SA, and Ca²⁺ signaling. As a chloroplast-localized protein, CaS participates in Ca²⁺-signaling pathways. However, the mechanisms underlying CaS-mediated immune regulation and whether to be attacked by pathogens remain unclear. Here, we created AcCaS-overexpressing transgenic plants, then we found that AcCaS activates chloroplast reactive oxygen species (ROS) bursts and enhances resistance against Pseudomonas syringae pv. actinidiae (Psa). Mutational analysis revealed that the chloroplast transit peptide (cTP) of AcCaS is essential for its immune function, and deletion of cTP abolished ROS production and disease resistance. Yeast two-hybrid reveals that Psa employs the effector HopAU1 targets AcCaS in kiwifruit. Luciferase complementation imaging, and microscale thermophoresis assays identified Asn-121 of AcCaS as the critical residue mediating both HopAU1 binding and Ca²⁺ sensing. Strikingly, molecular modeling and competitive binding experiments showed that HopAU1 directly occupies the Ca²⁺-binding site at Asn-121, thereby blocking calcium signaling and suppressing chloroplast immunity. In summary, this study uncovers that AcCaS enhances resistance against Psa by activating chloroplast ROS and binding with Ca²⁺. The Asn-121 residue plays a pivotal role in Ca²⁺-binding and HopAU1-mediated immune suppression, as mutations at this site abolish both activities. These findings revealed the battle of chloroplast Ca² signaling in plant-pathogen conflicts and provide a mechanistic basis for engineering AcCaS-centered resistance in kiwifruit.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China grant 32102174 awarded to W.L. and the National Key R&D Program of China (2022YFD1400200) to L.H. We are deeply grateful to Dr. Jinlong Zhang for his generous support and invaluable guidance throughout this study. Special thanks are extended to Dr. Zhiyuan Yin from Nanjing Agricultural University for his critical review and constructive suggestions on the manuscript. We also wish to acknowledge Prof. Yu Du and Prof. Cong Jiang at Northwest A&F University for their insightful discussions and expert advice.

Author contributions

R.L., W.L., and L.L.H. conceived the study. R.L. and Y.L.Z. performed the experiments. X.F.D. collected samples. X.X.W. and R.L. conducted data analysis. R.L. and Y.L.Z. wrote the manuscript with contributions from all authors. Professor L.L.H and W.L. supervised the experiment and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Data availability

The raw RNA-Seq data that support the findings of this study are openly available in NCBI at (https://www.ncbinlm.nih.gov/sra/PRINA1054973), reference number (PRJNA1054973). The sequence data in this study can be found in the Hong Yang v3 genome (https://kiwifruitgenome.atcgn.com) under accession numbers in the article and Supporting Information Tables S1. All the relevant data supporting the findings of this study are available in the article and supplementary data.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Li Y, Zhang D, Wang X. et al. LACCASE35 enhances lignification and resistance against Pseudomonas syringae pv. Actinidiae infec-tion in kiwifruit. Plant Physiol. 2025;197:kiaf040

[2] Yue J, Chen Q, Wang Y. et al. Telomere-to-telomere and gap-free reference genome assembly of the kiwifruit Actinidia chinensis. Hortic Res. 2023;10:uhac264

[3] Vanneste JL, Poliakoff F, Audusseau C. et al. First report of Pseudomonas syringae pv. Actinidiae, the causal agent of bacterial canker of kiwifruit in France. Plant Dis. 2011; 95:1311-1

[4] Chen H, Hu Y, Qin K. et al. A serological approach for the identification of the effector hopz5 of Pseudomonas syringae pv. Actinidiae: a tool for the rapid immunodetection of kiwifruit bacterial canker. J Plant Pathol. 2018; 100:171-7

[5] Kim M-J, Chae D-H, Cho G. et al. Characterization of antibacterial strains against kiwifruit bacterial canker pathogen. Plant Pathol J. 2019; 35:473-85

[6] Vanneste JL. The scientific, economic, and social impacts of the New Zealand outbreak of bacterial canker of kiwifruit (Pseu-domonas syringae pv. Actinidiae). Annu Rev Phytopathol. 2017; 55: 377-99

[7] Williams H, Ting C, Nejati M. et al. Improvements to and large-scale evaluation of a robotic kiwifruit harvester. JField Robot. 2020; 37:187-201

[8] Liu J, Gong P, Lu R. et al. Chloroplast immunity: a cornerstone of plant defense. Mol Plant. 2024; 17:686-8

[9] Zurbriggen MD, Carrillo N, Tognetti VB. et al. Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-host interaction between tobacco and Xanthomonas campestris pv. Vesicatoria. Plant J. 2009; 60:962-73

[10] Jelenska J, Yao N, Vinatzer BA. et al. A J domain virulence effector of Pseudomonas syringae remodels host chloroplasts and sup-presses defenses. Curr Biol. 2007; 17:499-508

[11] López-Solanilla E, Bronstein PA, Schneider AR. et al. HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant inter-actions. Mol Microbiol. 2004; 54:353-65

[12] Rodríguez-Herva JJ, González-Melendi P, Cuartas-Lanza R. et al. A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses: P. Syringae HopN1 targets photosynthesis. Cell Microbiol. 2012; 14: 669-81

[13] Li G, Froehlich JE, Elowsky C. et al. Distinct Pseudomonas type-III effectors use a cleavable transit peptide to target chloroplasts. Plant J. 2014; 77:310-21

[14] LiuR, ChenT, Yin X. et al. A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS production in grapevine. Plant J. 2021; 106:1557-70

[15] Han S, Tang R, Anderson LK. et al. A cell surface receptor medi-ates extracellular Ca2+ sensing in guard cells. Nature. 2003; 425: 196-200

[16] Xu Y, Huang B. Comparative transcriptomic analysis reveals common molecular factors responsive to heat and drought stress in Agrostis stolonifera. Sci Rep. 2018; 8:15181

[17] Zhao X, Xu M, Wei R. et al. Expression of OsCAS (calcium-sensing receptor) in an Arabidopsis mutant increases drought tolerance. PLoS One. 2015; 10:e0131272

[18] Wang L, Yamano T, Kajikawa M. et al. Isolation and character-ization of novel high-CO2-requiring mutants of Chlamydomonas reinhardtii. Photosynth Res. 2014; 121:175-84

[19] Wang L, Yamano T, Takane S. et al. Chloroplast-mediated regula-tion of CO2 -concentrating mechanism by Ca 2+-binding protein CAS in the green alga Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 2016; 113:12586-91

[20] Huang S, Chen J, Dong X. et al. Calcium and calcium recep-tor CAS promote Arabidopsis thaliana de-etiolation. Physiol Plant. 2012; 144:73-82

[21] Li B, Hou L, Song C. et al. Biological function of calcium-sensing receptor (CAS) and its coupling calcium signaling in plants. Plant Physiol Biochem. 2022; 180:74-80

[22] Nomura H, Komori T, Uemura S. et al. Chloroplast-mediated acti-vation of plant immune signalling in Arabidopsis. Nat Commun. 2012; 3:926

[23] Tang L, Yang G, Ma M. et al. An effector of a necrotrophic fungal pathogen targets the calcium-sensing receptor in chloro-plasts to inhibit host resistance. Mol Plant Pathol. 2020; 21: 686-701

[24] Zhao C, Liu W. et al. Two transcription factors, AcREM14 and AcC3H1, enhance the resistance of kiwifruit Actinidia chinensis var. chinensis to Pseudomonas syringae pv. Actinidiae. Hortic Res. 2024;11:uhad242

[25] Han P, Wang C, Li F. et al. Valsa Mali PR1-like protein modulates an apple valine-glutamine protein to suppress JA signaling-mediated immunity. Plant Physiol. 2024; 194:2755-70

[26] Zhang J, Zhou M, Liu W. et al. Pseudomonas syringae pv. Actinidiae effector HopAU1 interacts with calcium-sensing receptor to activate plant immunity. Int J Mol Sci. 2022; 23:508

[27] Zhao Z, Chen J, Gao X. et al. Comparative genomics reveal pathogenicity-related loci in Pseudomonas syringae pv. Actinidiae biovar 3. Mol Plant Pathol. 2019; 20:923-42

[28] Jones JDG, Dangl JL. The plant immune system. Nature. 2006; 444: 323-9

[29] Boller T, He SY. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science. 2009; 324:742-4

[30] Macho AP, Zumaquero A, Gonzalez-Plaza JJ. et al. Genetic anal-ysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. Phaseolicola. PLoS One. 2012; 7:e35871

[31] Tang R-J, Luan S. Regulation of calcium and magnesium home-ostasis in plants: from transporters to signaling network. Curr Opin Plant Biol. 2017; 39:97-105

[32] Perochon A, Aldon D, Galaud J-P. et al. Calmodulin and calmodulin-like proteins in plant calcium signaling. Biochimie. 2011; 93:2048-53

[33] Yeh S-M, Yoon M, Scott S. et al. NbPTR1 confers resistance against Pseudomonas syringae pv. Actinidiae in kiwifruit. Plant Cell Environ. 2024; 47:4101-15

[34] Zhou M, Zhang J, Zhao Z. et al. Pseudomonas syringae pv. Actinidiae unique effector HopZ5 interacts with GF14C to trigger plant immunity. Phytopathology. 2024; 114:2322-30

[35] Yuan M, Xin X-F. Bacterial infection and hypersensitive response assays in Arabidopsis-Pseudomonas syringae pathosystem. Bio Pro-toc. 2021; 11:e4268

[36] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001; 25:402-8

[37] Chen H, Zou Y, Shang Y. et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 2008; 146:323-4

[38] Nie J, Zhou W, Liu J. et al. A receptor-like protein from Nicotiana benthamiana mediates VmE02 PAMP-triggered immunity. New Phytol. 2021; 229:2260-72