Abstract
The receptor-like cytoplasmic kinase BIK1 and its close homologue PBL1 have been widely recognized as central components of plant immunity. However, most genetic studies of BIK1 and PBL1 functions were carried out with single transfer DNA (T-DNA) insertional mutant alleles. Some phenotypes observed in these mutants, for example autoimmunity, have been difficult to reconcile with the proposed role of BIK1 and PBL1 in pattern-triggered immunity. In this study, we generated several new alleles of bik1 and pbl1 by CRISPR–Cas9-based gene editing and systematically analysed these mutants alongside existing T-DNA insertional lines. These analyses reinforced the central role of BIK1 and PBL1 in pattern-triggered immunity mediated by both receptor kinases and receptor-like proteins. At the same time, however, we revealed several pleiotropic phenotypes associated with T-DNA insertions that are not necessarily linked to loss of BIK1 or PBL1 function. Further analyses of newly generated bik1 pbl1 double mutants uncovered an even greater contribution of these kinases to immune signalling and disease resistance than previously appreciated. These findings clarify longstanding ambiguities surrounding BIK1 and PBL1 functions.
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Data availability
The raw sequencing data of Col-0, bik1-1 and pbl1-1 generated by the DNBSEQ platform have been deposited in the National Center for Biotechnology Information database under the accession number PRJNA1255789. The third-generation raw data of bik1-1 have been submitted to the Genome Sequence Archive in the National Genomics Data Center with the record number CRA030180. The assembled genome is available via Figshare at https://doi.org/10.6084/m9.figshare.30148612.v1 (ref. 58). Other data are provided in Supplementary Table 1. Materials created in this study are available from the laboratories of the corresponding authors. Source data are provided with this paper.
Change history
04 March 2026
A Correction to this paper has been published: https://doi.org/10.1038/s41477-026-02259-y
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Acknowledgements
We thank S. Han for sharing GCaMP6m transgenic line and S. Yang for original Col-0. The study was supported by Biological Breeding—National Science and Technology Major Projects (2023ZD04070) to M.H., National Natural Science Foundation of China (31761143017), National Key R&D Program of China (2021YFA1300700) and Yazhouwan National Laboratory Project (2310JM01) to J.-M.Z., funding from University of Zurich, European Research Council under the grant agreement (nos. 309858 and 773153, grants ‘PHOSPHOinnATE’ and ‘IMMUNO-PEPTALK’), Swiss National Science Foundation (grants nos. 31003A_182625 and 310030_212382) to C.Z., Collaborative Research Center funding from the German Research Foundation (grant no. CRC1101-D10) to T.N. and funding from University of Michigan to P.H. and L.S.
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Conceptualization: J.-M.Z., C.Z. and P.H. Methodology: B.S., S.C., L.K., Y.G. and S.M. Investigation: B.S., S.C., L.K., S.-I.K., J.F., X.L., M.L., Y.Z., H.W. and M.H. Visualization: B.S., S.C., H.W., Y.G. and S.M. Funding acquisition: J.-M.Z., C.Z., P.H., L.S. and T.N. Project administration: J.-M.Z., C.Z. and P.H. Supervision: J.-M.Z., C.Z., P.H., L.S. and T.N. Writing: J.-M.Z., C.Z., P.H., T.N., T.A.D., H.W., B.S. and S.M.
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Extended data
Extended Data Fig. 1 Alleles of bik1 generated by CRISPR and T-DNA insertions.
a, Diagram shows the nature of mutations of various bik1 alleles. Note that the bik1-1 allele carries a 4.3 Mb chr 1 fragment immediately before the T-DNA insertion. For CRISPR-Cas9 alleles, red numbers in parentheses indicate number of nucleotides deleted or inserted into BIK1. Graph on the left depicts predicted BIK1 polypeptide length of each allele. Note each of the bik1-1Δ/2 and bik1-6/8 alleles was obtained from two independent events. b, Deletion of the truncated coding region from bik1-1 (bik1-1Δ) failed to rescue the growth phenotype (recorded in Beijing). Scale bar: 1 cm. c, Immunoblot of selected bik1 alleles using anti-BIK1 antibodies (Ann Arbor). Total protein isolated from indicated seedlings was examined with anti-BIK1 immunoblot. Ponceau (Ponc) staining of Rubisco (RBC) indicates equal loading of protein. d, Growth phenotypes of different bik1 alleles recorded in Tübingen. Scale bar: 2 cm. e, Primary root length of bik1 alleles under normal and Pi deficient conditions (Ann Arbor). Seedlings were grown in 1/2 (Mock) for 7 days then transferred to 1/2 MS without phosphate (- Pi) or 250 μM KH2PO4 (+ Pi) for another 7 days. f, Rosette width of bik1 alleles relative to Col-0 on 1/2 MS (mock) or 1/2 MS supplemented with mannitol (Ann Arbor). Seedlings were grown in 1/2 (Mock) or 1/2 MS + 100 mM mannitol for 15 days. Violin plots in (e) and (f) show individual data points (e, n = 12-26, f, n = 29-33) and median values (centre lines). **P ≤ 0.01, ****P ≤ 0.0001 (two-way ANOVA, Tuckey’s post-test). ns, not significant. Each experiment was repeated twice with consistent results, and data from one representative experiment are shown.
Extended Data Fig. 2 BIK1 positively regulates immune responses to different immunogenic patterns.
a-c, The bik1-1, bik1-2 and bik1-3 mutants all show impaired ROS burst in response to flg22, Pep1, and chitin. 3 × pbl, pbl30/31/32 triple mutant. d-g, The BIK1 transgene fully restores Pep1- and flg22-induced ROS burst to bik1-3. Two independent transgenic lines (#21 and #32) of bik1-3 complemented with BIK1 under the control of the native promoter were tested. h, bik1-1 shows elevated whereas bik1-2 and bik1-3 show reduced ROS burst in response to nlp20. i, bik1-1, but not bik1-2 and bik1-3, shows enhanced resistance to Pst infiltrated into the leaf. The experiments in (a, h) were performed in Tübingen. All other experiments were performed in Beijing and Zurich. Data are total photo counts during the 40 min recording (a and h, n = 11-70), mean ± s.e.m. for real-time luminescence (n = 12 for b, 8 for c, g, 6 for d-f), or mean ± s.d. for bacterial titer (i, n = 8). Nine independent experiments were performed for (a and h), and data from all experiments are included, with different letters indicating significant differences at p ≤ 0.05 (Kruskal–Wallis test, Dunn’s multiple comparison test). The centre line indicates the median, the bounds of the box show the 25th and the 75th percentiles, and the whiskers indicate 1.5 × IQR. Experiments in (b-g) were repeated ≥ three times with similar results, and data from one experiment are shown. (i) shows representative data from three experiments in which bik1-2, bik1-3 and Col-0 did not differ. See Fig. 3b. **P ≤ 0.01 (one-way ANOVA, Tukey’s post-test). ns, not significant.
Extended Data Fig. 3 Analyses of the pbl1-1 line and generation of gene-edited pbl1 alleles.
a, b, Lack of PR1 induction in response to Pst (a) and BTH (b) in the pbl1-1 mutant. c, The lack of PR1 induction was not restored in pbl1-1 stable transgenic lines complemented with PBL1 under the control of the 35S promoter. d, Ethylmethane sulfonate mutant alleles of pbl1-2 and pbl1-5 show normal PR1 induction in response to BTH. e, The pbl1-1 line carries a previously unknown T-DNA insertion in the PR1 promoter. f, Diagram shows the nature of mutations of various pbl1 alleles. Red numbers in parentheses indicate number of nucleotides deleted or inserted into PBL1. Graph on the left depicts predicted PBL1 polypeptide length of each allele. The original pbl1-1 line contains a second T-DNA insertion in the promoter of PR1. Note we named the ethylmethane sulfonate alleles of pbl1 (cce5-1 through cce5-4)8, pbl1-2 through pbl1-6. g, Growth phenotype of bik1-9, pbl1-9, and bik1-10 pbl1-10 (recorded in Zurich). Scale bar: 2 cm. Data are mean ± s.d. ****P ≤ 0.0001 (one-way ANOVA, Tukey’s post-test, n = 3). ns, not significant. Each experiment was repeated at least three times with similar results, and data from one representative experiment are shown.
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Song, B., Choi, S., Kong, L. et al. New alleles of Arabidopsis BIK1 reinforce its predominant role in pattern-triggered immunity and caution interpretations of other reported functions. Nat. Plants 12, 284–293 (2026). https://doi.org/10.1038/s41477-025-02187-3
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DOI: https://doi.org/10.1038/s41477-025-02187-3
