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Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases

Abstract

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development and immunity in plants, but the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLAGELLIN SENSING 2 (FLS2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We surprisingly found that the loss of the BSU1 family phosphatases activates effector-triggered immunity and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine 251. Mutation of serine 251 reduces BSU1’s ability to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress effector-triggered immunity and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine 764 upon brassinosteroid signalling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid–BRI1 and flagellin–FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signalling specificities for diverse plant receptor kinases.

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Fig. 1: The bsu-q mutant shows enhanced ETI and defective flagellin signalling.
Fig. 2: BSU1 interacts with BIK1 and MEKK1.
Fig. 3: Flagellin induces phosphorylation of BSU1 at Ser251.
Fig. 4: BIK1 phosphorylates BSU1 at Ser251.
Fig. 5: Phosphorylation of BSU1 Ser251 is required for MAPK activation and immunity but not for BR signalling.

Data availability

The RNA-seq data that support the findings of this study have been deposited in GEO with the accession code GSE140037. The MS proteomics data are available via ProteomeXchange with the identifiers PXD016283 (username, reviewer67695@ebi.ac.uk; password, pCDR9H2R, for DDA mode) and PXD016257 (username, reviewer88035@ebi.ac.uk; password, IQKH8jha, for PRM mode). Source data are provided with this paper.

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Acknowledgements

We thank J.-M. Zhou for sharing the BIK1::BIK1–HA and MEKK1–FLAG seeds, and L. Shan and P. He for the GST–BIK1 and GST–BIK1Km constructs. We thank A. V. Reyes for generating the bee swarm box plot figures. This research was supported by grants from the NIH (no. R01GM066258 to Z.-Y.W. and no. R01GM135706 to S.-L.X.), the National Research Foundation of Korea funded by the Ministry of Science, ICT, Future Planning (nos NRF-2021R1A2C1006617 and 2020R1A6A1A06046728 to T.-W.K. and no. 2021R1A2C1007516 to S.-K.K.), the NSF (no. NSF-IOS 2026368 to M.B.M.), the Howard Hughes Medical Institute and NIH (no. P41GM103481 to A.L.B.), and the Carnegie Institution for Science endowment fund to the Carnegie MS facility.

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Authors and Affiliations

Authors

Contributions

C.H.P., Y.B., T.-W.K. and Z.-Y.W. designed the research. C.H.P. and Y.B. performed most of the experiments. C.H.P. and Y.B. performed the PAMP treatment and immunoblotting. C.H.P. performed RNA-seq. C.H.P. and Y.B. analysed the RNA-seq data. C.H.P., Y.B., J.-H.Y., S.-H.K., S.-K.K. and T.-W.K. performed and analysed the yeast two-hybrid assay and in vitro pull-down assay. S.-H.K., S.-K.K. and T.-W.K. performed the SA quantification. C.H.P., S.-L.X., A.L.B. and R.S. performed the MS analysis. C.H.P., J.-G.K. and M.B.M. performed and analysed the bacterial growth assay. C.H.P., Y.B. and N.Y.X. performed the immunoblot and seedling flood inoculation assay. C.H.P., Y.B., S.-K.K., T.-W.K., M.B.M. and Z.-Y.W. wrote the manuscript.

Corresponding authors

Correspondence to Seong-Ki Kim, Tae-Wuk Kim or Zhi-Yong Wang.

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Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Gene ontology (GO) analysis of genes differentially expressed in bsu-q.

(a, b) Enrichment of GO annotation in categories of biological process for genes increased (a) and decreased (b) in bsu-q. Blue and orange columns indicate the percent of genes of input (Supplementary Table 2) and reference (TAIR10), respectively. Bold with underlines indicate enriched GO categories of biological process.

Extended Data Fig. 2 Expression of flagellin-induced genes are altered in bsu-q.

(a, b) The bsu-q mutant has defects in flg22-induced gene expression. Heat map (a) and scatter plot (b) of log2 fold change (FC, flg22/mock) values of flg22-responsive genes in wild type (WT) and bsu-q from RNA-seq data. (c) Quantitative RT-PCR of flg22-induced FRK1, At2g17740, ROF2 and CRK13 expression in WT and bsu-q. The relative expression levels are normalized to the PP2A gene expression and to the wild type sample. Asterisks indicate statistically significant difference between mock and flg22 treated samples (two-tail t-test, p = 6.3014E-7, 0.02168, 0.002650, 0.0006797, 1.9289E-5 and 0.003883 from left to right). The graph shows average expression level ± SEM of six biological repeats for FRK1 (n = 6) and three biological repeats for other genes (n = 3).

Source data

Extended Data Fig. 3 Loss of BSU1 family impairs flg22 induction of ROS and increases SA level.

(a) Flg22-induced ROS burst in WT and bsu-q. are indicated. Each data point represents the average of relative light unit (RLU) ± SEM of at least 12 replicates from 2 biological repeats (6 samples for each repeat). Each repeat showed similar results. (b) The bsu-i mutant accumulates increased levels of salicylic acid. The SA levels (microgram per gram fresh tissue) of wild type and bsu-i were measured from 0.1 g of 11-day-old seedlings grown on 10 µM estradiol in two biological repeats.

Source data

Extended Data Fig. 4 The flg22- and elf18-induced MPK3/6 phosphorylation in the BSU1 family mutants.

(a) Flg22-induced MPK3/6 phosphorylation in wild-type (WT) and bsl2;bsl3. Ten-day-old seedlings were treated with 100 nM flg22 for 10 min. Immunoblot was performed using anti-phospho-p44/42 MAPK and anti-BSL2/3 antibodies. Ponceau S staining shows equal protein loading. (b) Elf18-induced MPK3/6 phosphorylation in bsu-i. Twelve-day-old seedlings grown on 20 µM estradiol containing media were treated with 1 µM elf18 for 10 min. Immunoblot was performed using anti-phospho-p44/42 MAPK and anti-BSL2/3 antibodies. Ponceau S staining shows equal protein loading.

Source data

Extended Data Fig. 5 Yeast-two-hybrid assays for interactions between BIK1 family RLCKs and BSU1/BSLs proteins.

Yeast-two-hybrid assay for interaction between BIK1/PBLs and BSU1/BSLs proteins. Yeast cells co-expressing AD-BIK1/PBLs and BD-BSU1/BSLs were grown on synthetic dropout medium containing 2.5 mM 3-amino-1, 2, 4-triazole (3AT).

Extended Data Fig. 6 SILIA IP-MS PRM analysis of flg22-induced BSU1 phosphorylation.

(a) Chromatograms of fragment ions from the replicate SILIA IP-MS PRM experiment of Fig. 3c using reciprocal labeling. (b) The ratio of phosphorylated and non-phosphorylated MDSDNVWTPVPAVAPpSPR peptides. The values of peak area were used for quantification. The ratios of S251 phosphorylation between flg22-treated (+flg22) and mock-treated (-flg22) samples are shown.

Extended Data Fig. 7 Alignment of sequence around S251 of BSU1 with those of paralogs.

Amino acid sequences of the indicated species were obtained from Maselli et al., 2014 (22). Asterisks indicate BSU1 S251.

Extended Data Fig. 8 Relative band intensity of Fig. 5a, c.

(a) Relative band intensities of immunoblot images of flg22-treated samples using anti-phospho-p44/42 MAPK antibody (anti-p-MPK) in Fig. 5a were measured by ImageJ. The band intensity of each bsu-i sample was set to 1. The graph shows the average band intensity ± SEM from three repeat experiments. One-tail t-tests show that the band intensities of BSU1;i-1 and BSU1;i-2 are significantly stronger than that of S251A;i (p = 0.0268 and p = 0.0157). (b) Band intensities of immunoblot images, represented by Fig. 5c, were measured by ImageJ. The band intensity of GST-BIN2 was divided by that of MBP-BSU1, and then the ratio between +GST-CDG1 and -GST-CDG1 is presented to show the effect of CDG1 on BSU1-BIN2 binding. The graph shows the average binding activity ± SEM from three repeat experiments. One-tail t-test shows that the binding activity of S251A is significantly higher than that of S764A. (p = 0.041983).

Source data

Extended Data Fig. 9 BSU1 protein level of seedlings in Fig. 5d, e.

Total protein was extracted from 7-day old light-grown seedlings. BSU1-YFP protein level was analyzed by immunoblotting using anti-GFP antibody. Ponceau S staining image shows equal protein loading. These transgenic lines were used in Fig. 5d, e.

Source data

Supplementary information

Reporting Summary

Supplementary Data 1

Supplementary Tables 1–6.

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Extended Data Fig. 8

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Extended Data Fig. 9

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Park, C.H., Bi, Y., Youn, JH. et al. Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases. Nat. Plants 8, 646–655 (2022). https://doi.org/10.1038/s41477-022-01167-1

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