A Ca²⁺ sensor BraCBL1.2 involves in BraCRa-mediated clubroot resistance in Chinese cabbage

Abstract

Clubroot disease caused by Plasmodiophora brassicae (P. brassicae) severely threatens the cultivation of Cruciferous plants, especially Chinese cabbage. Recently, resistance genes in plants have been reported to encode for a Ca²⁺-permeable channel in the plasma membrane, which can mediate the cytosolic Ca²⁺ increase in plant cells upon pathogen attack. However, the downstream Ca²⁺ sensor and decoder are still unknown. In this study, we identified the virulent and avirulent P. brassicae isolates (Pbs) of two near isogenic lines, CR 3-2 and CS 3-2, with CR 3-2 harboring clubroot resistant gene BraCRa. The transcriptomic analysis was then conducted with CR 3-2 after inoculating with virulent isolate PbE and avirulent isolate Pb4. From the differentially expressed genes of transcriptomic data, we identified a Ca²⁺-sensor encoding gene, BraCBL1.2, that was highly induced in CR 3-2 during infection by Pb4 but not by PbE. Moreover, GUS histochemical staining and subcellular localization analysis revealed that BraCBL1.2 was specifically expressed in the root hair cells of Arabidopsis and encoded a putative Ca²⁺ sensor localized in the plasma membrane. We also developed an assay to investigate the BraCRa-mediated hypersensitive response (HR) in tobacco leaves. The results suggest that BraCBL1.2 is involved in the BraCRa-mediated plant ETI immune response against P. brassicae. In addition, we verified that overexpression of BraCBL1.2 enhanced clubroot resistance in Arabidopsis. Collectively, our data identified the involvement of a Ca²⁺ sensor in BraCRa-mediated clubroot resistance in Chinese cabbage, providing a theoretical basis for further research on the resistance of Chinese cabbage to P. brassicae.

References

[1.] Liu L, Qin L, Zhou Z. et al. Refining the life cycle of Plasmodiophora brassicae. Phytopathology. 2020;110:1704-12

[2.] Dixon GR. The occurrence and economic impact of Plasmodio-phora brassicae and clubroot disease. J Plant Growth Regul. 2009;28: 194-202

[3.] Schwelm A, Fogelqvist J, Knaust A. et al. The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases. Sci Rep. 2015;5:11153

[4.] Peng G, Lahlali R, Hwang S-F. et al. Crop rotation, cultivar resistance, and fungicides/biofungicides for managing clubroot (Plasmodiophora brassicae) on canola. Can J Plant Pathol. 2014;36: 99-112

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

[6.] Muthamilarasan M, Prasad M. Plant innate immunity: an updated insight into defense mechanism. J Biosci. 2013;38:433-49

[7.] Jacob P, Kim NH, Wu F. et al. Plant "helper" immune recep-tors are Ca(2+)-permeable nonselective cation channels. Science. 2021;373:420-5

[8.] Ueno H, Matsumoto E, Aruga D. et al. Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa. Plant Mol Biol. 2012;80:621-9

[9.] Hatakeyama K, Suwabe K, Tomita RN. et al. Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS One. 2013;8:e54745

[10.] Hashimoto K, Kudla J. Calcium decoding mechanisms in plants. Biochimie. 2011;93:2054-9

[11.] Tian W, Hou C, Ren Z. et al. A calmodulin-gated calcium channel links pathogen patterns to plant immunity. Nature. 2019;572: 131-5

[12.] Bi G, Su M, Li N. et al. The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling. Cell. 2021;184:3528-3541.e12

[13.] Wang W, Qin L, Zhang W. et al. WeiTsing, a pericycle-expressed ion channel, safeguards the stele to confer clubroot resistance. Cell. 2023;186:2656-2671.e18

[14.] Dodd AN, Kudla J, Sanders D. The language of calcium signaling. Annu Rev Plant Biol. 2010;61:593-620

[15.] Kudla J, Xu Q, Harter K. et al. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci U S A. 1999;96:4718-23

[16.] Wu P, Wang W, Duan W. et al. Comprehensive analysis of the CDPK-SnRK superfamily genes in Chinese cabbage and its evo-lutionary implications in plants. Front Plant Sci. 2017;8:162

[17.] Jung HJ, Kayum MA, Thamilarasan SK. et al. Molecular charac-terisation and expression profiling of calcineurin B-like (CBL) genes in Chinese cabbage under abiotic stresses. Funct Plant Biol. 2017;44:739-50

[18.] Guo N, Wang G, Zong M. et al. Genome-wide identification, and phylogenetic and expression profiling analyses of CaM and CML genes in Brassica rapa and Brassica oleracea. Gene. 2018;677: 232-44

[19.] Kim NH, Jacob P, Dangl JL. Con-Ca(2+) -tenating plant immune responses via calcium-permeable cation channels. New Phytol. 2022;234:813-8

[20.] Luan S, Wang C. Calcium signaling mechanisms across king-doms. Annu Rev Cell Dev Biol. 2021;37:311-40

[21.] Gao X, Chen X, Lin W. et al. Bifurcation of Arabidopsis NLR immune signaling via Ca²⁺-dependent protein kinases. PLoS Pathog. 2013;9:e1003127

[22.] Lu Y-J, Li P, Shimono M. et al. Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunity. Nat Commun. 2020;11:6234

[23.] Liu P, Duan Y, Liu C. et al. The calcium sensor TaCBL4 and its interacting protein TaCIPK5 are required for wheat resistance to stripe rust fungus. JExp Bot. 2018;69:4443-57

[24.] de la Torre F, Gutierrez-Beltran E, Pareja-Jaime Y. et al. The tomato calcium sensor Cbl10 and its interacting protein kinase Cipk6 define a signaling pathway in plant immunity. Plant Cell. 2013;25:2748-64

[25.] Yang S, Li J, Lu J. et al. Potato calcineurin B-like protein CBL4, interacting with calcineurin B-like protein-interacting protein kinase CIPK2, positively regulates plant resistance to stem canker caused by Rhizoctonia solani. Front Microbiol. 2023;13:1032900

[26.] Hwang SF, Ahmed HU, Zhou Q. et al. Influence of resistant cultivars and crop intervals on clubroot of canola. Can J Plant Sci. 2019;99:862-72

[27.] Pang W, Liang Y, Zhan Z. et al. Development of a sinitic clubroot differential set for the pathotype classification of Plasmodiophora brassicae. Front Plant Sci. 2020;11:568771

[28.] Batistič O, Kudla J. Plant calcineurin B-like proteins and their interacting protein kinases. Biochim Biophys Acta. 2009;1793: 985-92

[29.] Yin X, Wang Q, Chen Q. et al. Genome-wide identification and functional analysis of the calcineurin B-like protein and cal-cineurin B-like protein-interacting protein kinase gene families in turnip (Brassica rapa var. rapa). Front Plant Sci. 2017;8:1191

[30.] Chen C, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13:1194-202

[31.] Batistič O, Sorek N, Schültke S. et al. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca2+ signaling complexes in Arabidopsis. Plant Cell. 2008;20:1346-62

[32.] Kageyama K, Asano T. Life cycle of Plasmodiophora brassicae. J Plant Growth Regul. 2009;28:203-11

[33.] Xu L, Yang H, Ren L. et al. Jasmonic acid-mediated aliphatic Glucosinolate metabolism is involved in clubroot disease development in Brassica napus L. Front Plant Sci. 2018;9: 750

[34.] Tsitsigiannis DI, Keller NP. Oxylipins as developmental and host-fungal communication signals. Trends Microbiol. 2007;15: 109-18

[35.] Dave A, Graham IA. Oxylipin signaling: a distinct role for the Jasmonic acid precursor cis-(+)-12-Oxo-Phytodienoic acid (cis-OPDA). Front Plant Sci. 2012;3:42

[36.] Zamani-Noor N, Hornbacher J, Comel CJ. et al. Variation of glu-cosinolate contents in clubroot-resistant and -susceptible Bras-sica napus cultivars in response to virulence of Plasmodiophora brassicae. Pathogens. 2021;10: 563.

[37.] Ludwig-Müller J. Glucosinolates and the clubroot disease: defense compounds or auxin precursors? Phytochem Rev. 2009;8: 135-48

[38.] Zhou Z, Bi G, Zhou JM. Luciferase complementation assay for protein-protein interactions in plants. Curr Protoc Plant Biol. 2018;3:42-50