Abstract
Rapid production of H2O2 is a hallmark of plant responses to diverse pathogens and plays a crucial role in signalling downstream of various receptors that perceive immunogenic patterns. However, mechanisms by which plants sense H2O2 to regulate immunity remain poorly understood. We show that endogenous H2O2 generated upon immune activation is sensed by the thiol peroxidase PRXIIB via oxidation at Cys51, and this is essential for stomatal immunity against Pseudomonas syringae. We further show that in immune-stimulated cells, PRXIIB conjugates via Cys51 with the type 2C protein phosphatase ABA insensitive 2 (ABI2), subsequently transducing H2O2 signal to ABI2. This oxidation dramatically sensitizes H2O2-mediated inhibition of the ABI2 phosphatase activity in vitro and is required for stomatal immunity in plants. Together, our results illustrate a redox relay, with PRXIIB as a sensor for H2O2 and ABI2 as a target protein, that mediates reactive oxygen species signalling during plant immunity.
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All data to support the conclusions of this manuscript are provided in the main figures, Extended Data figures and supplementary information. Source data are also provided in supplementary information.
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Acknowledgements
We thank Z. Gong for providing triple mutant abi1 abi2 hab1. This work was supported by the National Natural Science Foundation of China (31830019, 3211001023 and 31521001 to J.-M.Z.; 21922702, 81973279 and 31770885 to J.Y.; 32170288 and 31900222 to G.B.), the National Key R&D Program of China (2016YFA0501303 to J.Y.), the State Key Laboratory of Plant Genomics (SKLPG2016B-2 to J.-M.Z.), the Strategic Priority Research Program of the CAS (XDPB160201 to J.-M.Z.), the Hainan Excellent Talent Team (to J.-M.Z.) and the State Key Laboratory of Proteomics (SKLP-K201703 and SKLP-K201804 to J.Y.).
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J.-M.Z., J.Y. and G.B. designed the research. M.H. and G.B. performed the majority of the experiments, assisted by X.Z. L.F. contributed to chemoproteomic and MS analyses. J.-M.Z., J.Y., J.Z., J.L. and G.B. wrote the manuscript with comments from all authors.
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Extended data
Extended Data Fig. 1 Gene expression profiles and localization of TPXs.
Shown is a heat map of transcript levels of the indicated genes at different developmental stages obtained from Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Color bar represents log10 expression values, thereby blue, white and red represents lower, medium and higher expression levels, respectively. The predicted or experimentally supported subcellular localization is shown on the right.
Extended Data Fig. 2 PRXIIs and GPXs are required for stomatal immunity. Gene editing of peroxidases.
a, Mutations of GPX2, GPX5, and GPX6 in the gpx2 gpx5 gpx6 (gpx2-2 gpx5-1 gpx6-3) triple mutant generated by CRISPR-Cas9. b, Mutations in the prxIIB-1 single mutant and prxIIB prxIIC prxIID (prxIIB-1 prxIIC-1 prxIID-1) triple mutant generated by CRISPR-Cas9. Nucleotides in red indicate single base insertion in the mutants. c, The prxIIB single mutant and prxIIB prxIIC prxIID triple mutant are impaired in the restriction of bacterial entry. This experiment was repeated three times with similar results. d, Accumulation of PRXIIB in prxIIB and prxIIB prxIIC prxIID plants. PRXIIB protein was detected by immunoblotting with anti-PRXIIB antibodies. This experiment was repeated three times with similar results. e, PRXIIB restores stomatal immunity to the prxIIB prxIIC prxIID triple mutant. f, The gpx2 gpx5 gpx6 mutant is mildly affected in the restriction of bacterial entry. For experiments in c, e, and f, leaf discs of indicated plants were soaked with Pst DC3000 cor − for 1.5 h, and then the bacterial number was determined. Different letters indicate significant difference at P < 0.05. Data are mean ± sd (n = 12, one-way ANOVA, Tukey post-test). Each experiment was repeated at least three times with similar results.
Extended Data Fig. 3 The flg22-triggered accumulation of H2O2 in the apoplast and cytosol occur normally in the gpx2 gpx5 gpx6 and prxIIB prxIIC prxIID mutants.
a, Flg22 induces normal ROS burst in the apoplast of the gpx2 gpx5 gpx6 and prxIIB prxIIC prxIID mutants. Leaf strips of four-week-old plants of the indicated genotypes were incubated in H2O overnight. One µM flg22 was added, and the production of apoplastic H2O2 was measured immediately by the luminol-based assay. Relative amounts of H2O2 are shown as relative luminescence units. Each data point is presented as mean ± sd, n = 10. b, The flg22-induced increase of H2O2 in the cytosol is not affected in the gpx2 gpx5 gpx6 and prxIIB prxIIC prxIID mutants. Leaf discs of four-week-old plants of transgenic lines carrying the roGFP2-Orp1 reporter gene in the indicated genetic background were incubated in H2O overnight, placed in a luminometer, sequentially excited at 405 nm and 488 nm, and emission was recorded at 510 nm. One µM flg22 was added at the indicated time point during the recording. The 405 nm/488 nm excitation ratios provide an indication for relative amounts of H2O2 in the cytosol. Each data point is presented as mean ± sd, n = 10. Each experiment was repeated three times with similar results.
Extended Data Fig. 4 Cys51 of PRXIIB is sufenylated in response to flg22 and required for ABA-induced stomatal closure.
a, Flg22 induces sulfenylation on PRXIIB Cys51. Col-0 protoplasts were treated with H2O or 1 µM flg22 for 20 min, and total protein was subjected to S-sulfenylome analysis. Cys-SOH mapped to Arabidopsis TPXs from flg22 and H2O treatments are shown in red and blue, respectively. flg22/H2O ratios were calculated and displayed with chromatograms. Note that no Cys-SOH was mapped to PRXIIC, PRXIID, and GPXs, which may be attributed to the low-abundance of the proteins (PRXIIC and PRXIID) and/or the stochastic nature of DDA (data-dependent acquisition)-based MS analysis. b, Accumulation of wild type PRXIIB-FLAG and PRXIIBC51S-FLAG proteins in T2 transgenic plants. Total protein from leaves was subjected to anti-FLAG immunoblot. This experiment was repeated three times with similar results. c, Cys51 of PRXIIB is essential for ABA-induced stomatal closure. Leaves of four-week old plants of the indicated genotypes were soaked with 100 µM ABA, and stomatal aperture was measured 2 h later. Different letters indicate significant difference at P < 0.05. Data are mean ± sd (n = 40, one-way ANOVA, Tukey post-test).
Extended Data Fig. 5 H2O2 induces the association of ABI2 with PRXIIB, GPX2, and APX1.
a. H2O2-induced PRXIIB-ABI2 association is dose-dependent. Protoplasts expressing PRXIIB-FLAG and ABI2-HA were treated with H2O2 at indicated concentrations, and CoIP was performed. The immunoprecipitates were eluted in the presence of 1 mM DTT. Quantification below the blot shows arbitrary densitometry units of CoIP products normalized to input ABI2-HA. Different letters indicate significant difference at P < 0.05. Data are mean of three independent experiments ± sem (one-way ANOVA, Tukey post-test). Note that the 5–20 µM H2O2 treatments had significantly more ABI2-HA compared to the no H2O2 control when student t- test was used. b, Exogenous H2O2 induces the association of ABI2 with PRXIIB, GPX2 and APX1. Protoplasts expressing indicated constructs were treated with 5 μM H2O2 for 10 min, and CoIP was performed. The immunoprecipitates were eluted in the presence of 1 mM DTT. Quantification below the blot shows arbitrary densitometry units of CoIP products normalized to input ABI2-HA. Different letters indicate significant difference at P < 0.05. Data are mean of three independent experiments ± sem (one-way ANOVA, Tukey post-test). c, H2O2 induces the formation of high molecular weight PRXIIB-ABI2 products that are sensitive to DTT. Shown are long exposures of the same immunoblots from Fig. 2b. Basal levels of PRXIIB-ABI2 conjugation are evident. Each experiment was repeated three times with similar results.
Extended Data Fig. 6 flg22 triggers ROS production in protoplasts in a manner dependent on FLS2 and RBOHD.
Protoplasts were prepared from the indicated plants, treated with 1 µM flg22, and ROS was measured immediately and expressed as relative luminescence units. Each data point represents mean ± sd, n ≥ 8 technical replicates (200 μl of protoplasts as one sample). This experiment was repeated three times with similar results.
Extended Data Fig. 7 Oxidation of PRXIIB and ABI2 in vitro.
a, b, H2O2 induces oxidation of PRXIIB, but not PRXIIBC51S. PRXIIB (a) and PRXIIBC51S (b) recombinant proteins were incubated with the indicated concentrations of H2O2, incubated for 10 min, and MAL-PEG 2000 was added to bind free thiols. The protein was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). The oxidation of thiols prevents binding by MAL-PEG 2000 and gives rise to smaller protein on SDS-PAGE. The apparent smaller size of PRXIIBC51S than the wild type PRXIIB in the reduced forms was caused by the lack of Cys51 thiol in the mutant protein which prevented binding of MAL-PEG 2000 to this site. c, ABI2 is not oxidized by H2O2 in vitro in the absence of PRXIIB. ABI2 recombinant protein was incubated with 5 μM H2O2 for the indicated times, and MAL-PEG 2000 was added to bind free thiols. The protein was subjected to SDS-PAGE and stained with CBB. Each experiment was repeated three times with similar results.
Extended Data Fig. 8 Disulfide bonds within the oxidized ABI2.
Shown are spectra of peptides corresponding to disulfide bonds within the oxidized ABI2 from Supplementary Table 2. The b and y ions are marked and displayed along the peptide sequence on top of the graph.
Extended Data Fig. 9 Cys residues of ABI2 involved in disulfide bonding are required for the association with PRXIIB.
a, ABI25CS abolishes the association with PRXIIB. Col-0 protoplasts expressing indicated constructs were treated with 100 μM H2O2 for 10 min, and CoIP was performed. The immunoprecipitates were eluted in the presence of 1 mM DTT and subjected to reducing gel and immunoblot. Quantification on the right shows arbitrary densitometry units of CoIP products normalized to input ABI2-HA. Different letters indicate significant difference at P < 0.05. Data are mean of three independent experiments ± sem (one-way ANOVA, Tukey post-test). b, ABI25CS abolishes the formation of H2O2-induced high molecular weight complex. Col-0 protoplasts expressing ABI2-HA or ABI25CS-HA were treated with 100 μM H2O2 for 10 min, and total protein was subjected into electrophoresis through a non-reducing gel. Each experiment was repeated three times with similar results.
Extended Data Fig. 10 PRXIIB Cys51, but not ABI2 oxidation, contributes to mesophyll immunity.
a, T2 plants of the indicated transgenic lines complemented with WT ABI2 and ABI25CS were analyzed for ABI2-HA protein accumulation by immunoblot. This experiment was repeated three times with similar results. b, c, PRXIIs are required for flg22-induced disease resistance in mesophyll cells in a manner dependent on Cys51. Plants were pre-infiltrated with H2O or flg22 one day before infiltration with Pst DC3000, and then the bacterial number was determined two days later. Different letters indicate significant difference at P < 0.05. Data are mean ± sd (n = 12, one-way ANOVA, Tukey post-test). d, Redox-relaying cysteines of ABI2 are not required for flg22-induced disease resistance in mesophyll cells. Plants were pre-infiltrated with H2O and flg22 one day before infiltration with Pst DC3000, and then the bacterial number was determined two days later. Different letters indicate significant difference at P < 0.05. Data are mean ± sd (n = 12, one-way ANOVA, Tukey post-test). Each experiment was repeated three times with similar results.
Supplementary information
Supplementary Table
Supplementary Table 1. Identification of PRXIIB-interacting proteins by LC–MS/MS. Supplementary Table 2. Identification of disulfide bonds on ABI2 and PRXIIB. Supplementary Table 3. Primers used in this study.
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Bi, G., Hu, M., Fu, L. et al. The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay. Nat. Plants 8, 1160–1175 (2022). https://doi.org/10.1038/s41477-022-01252-5
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DOI: https://doi.org/10.1038/s41477-022-01252-5
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