Extended Data Fig. 8: Ca²⁺ signalling by flg22 and elf26 is not affected in hpca1 mutants, and eH₂O₂ Ca²⁺ signalling is not affected in fls2, bak1 or ghr1 receptor kinase mutants.

a, b, Increases in [Ca²⁺]_i in wild-type and hpca1 mutant plotted as a function of increasing concentrations of flg22 (a) or elf26 (b). Data are mean ± s.d.; n = 32 seedlings. P = 0.172 (a), P = 0.062 (b), two way ANOVA. Experiments were performed as in Fig. 1f–h. c, e, Representative images of aequorin bioluminescence from the wild-type, hpca1 and fls2 seedlings, which were grown side-by-side and treated with 4 mM H₂O₂ (c) or 1 μM flg22 (e). d, f, Quantification of [Ca²⁺]_i in leaves from experiments in c or e is shown for treatment with H₂O₂ (d) or flg22 (f), treatment, respectively. Data are mean ± s.d. and representative of eight experiments; n = 16 seedlings. g, i, Representative aequorin bioluminescence images from wild-type, hpca1 and bak1 seedlings, which were grown side-by-side and treated with 4 mM H₂O₂ (g) or 1 μM flg22 (i). h, j, Quantification of [Ca²⁺]_i in leaves from experiments in g or i is shown for H₂O₂ (h) or flg22 treatment (j), respectively. Data are mean ± s.d. and representative of eight experiments; n = 16 seedlings. k, l, Representative aequorin bioluminescence images (k) and quantification of [Ca²⁺]_i increases (l) from wild-type, hpca1 and ghr1 seedlings, which were grown side-by-side and treated with 4 mM H₂O₂. Data in l are mean ± s.d. and representative of eight experiments; n = 16 seedlings. The receptors for MAMPs in innate immunity are commonly receptor kinases^6,26. The two best-characterized LRR-RKs, FLS2 and EFR, are receptors for flg22 and elf18–elf26, respectively²⁶. Both flg22 and elf18–elf26 induce [Ca²⁺]_i increases, which are abolished in their corresponding receptor mutants, fls2 and efr, respectively²⁶. We analysed whether HPCA1 could distinguish H₂O₂ produced in response to treatment with MAMPs. [Ca²⁺]_i increases triggered by flg22 or elf26 were not affected in hpca1 mutants. The mutants in FLS2 and its co-receptor BAK1 showed the defects in flg22-triggered [Ca²⁺]_i increases, but displayed wild-type level of eH₂O₂-induced [Ca²⁺]_i increases. The observation is consistent with the previous finding that the rbohD (NADPH oxidase D) mutant and treatment with the NADPH oxidase inhibitor diphenyleneiodonium do not affect the initial [Ca²⁺]_i increases triggered by 5-min treatment of flg22⁷⁶. GHR1 is an LRR-RK with an unknown ligand in the III subfamily (Extended Data Fig. 4a), and the ghr1 mutant displays several strong phenotypes in the eH₂O₂ and ABA signalling²⁸.

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