Abstract
The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1,2,3,4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants—fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants—are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.
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Data availability
The RNA-seq data have been deposited into the NCBI Gene Expression Omnibus under accession GSE142747. All data are available in the main text or the supplementary materials. Source data are provided with this paper.
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Acknowledgements
We thank Xin laboratory members for helpful discussions; the Greenhouse and Confocal Microscopy Imaging facilities at the CAS Center for Excellence in Molecular Plant Sciences for plant growth and technical support; G. Coaker from University of California, Davis for providing the RIN4 antibody; B. P. M. Ngou and P. Ding from J. Jones’ laboratory at the Sainsbury Laboratory for insightful discussions during manuscript preparation. This research was supported by the Chinese Academy of Sciences, Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology, National Key Laboratory of Plant Molecular Genetics and Chinese Academy of Sciences Strategic Priority Research Program (Type-B; project number: XDB27040211). G.B. was supported by the Youth Program of National Natural Science Foundation of China (NSFC) (project number: 31900222). The initial observation of PRR dependency for ETI resistance was made by X.-F.X. while at Michigan State University, supported by the US National Institute of General Medical Sciences (GM109928, to S.Y.H.).
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M.Y. and X.-F.X. conceptualized and designed experiments at the CAS Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology. M.Y. performed most experiments, including ROS detection, disease/HR assays, RNA-seq, and transcript and protein analysis. Z.J. and M.L. performed RIN4 cleavage, MAPK phosphorylation and gene-expression experiments. G.B., Y.W., M.Y. and B.C. performed protoplast experiments for detecting RBOHD phosphorylation. K.N. performed the disease assay and gene-expression analysis in bik1 and rbohd mutants. S.Y.H. and J.-M.Z. supervised K.N. and G.B., respectively. M.Y. and X.-F.X. wrote the paper and S.Y.H. and J.-M.Z. edited the paper.
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Extended data figures and tables
Extended Data Fig. 1 PRR/co-receptors are required for ETI elicited by different P. syringae avirulent effectors.
a, Pst DC3000 (avrRpt2) bacteria were infiltrated into Arabidopsis leaves at OD600 of 0.002 and populations were determined at 3 dpi (mean ± s.d.; n = 4 biologically independent samples, except n = 3 biologically independent samples for bbc-DC3000). Data were analysed using two-way ANOVA with Tukey’s test. b, AvrPphB- and AvrRps4-mediated ETI are also compromised in fec and bbc mutants. Plants were infiltrated with different strains at OD600 of 0.002. Bacterial populations were determined 3 dpi. Data were analysed using two-way ANOVA with Tukey’s test. (mean ± s.d.; n = 3 (Col-0 fec/bbc-DC3000(avrRpt2) and fec-DC3000(avrPphB)) or 4 (Col-0 fec/bbc-DC3000(avrRps4) and Col-0/bbc-DC3000(avrPphB)) biologically independent samples). c, Hypersensitive response was compromised in PRR/co-receptor mutants. Pst DC3000 (avrRpt2) bacteria were infiltrated at OD600 of 0.2 and images were taken about 7 h post infiltration (hpi). Experiments were repeated three times with similar trends.
Extended Data Fig. 2 RIN4 cleavage, transcript level of RPS2 and activation of MAPK cascades are not altered in the fec and bbc plants.
a, RIN4 cleavage in Col-0 and the PRR/co-receptor mutants after D36E or D36E(avrRpt2) inoculation. CBB, Coomassie blue staining. An equal amount of total protein was loaded in each lane. b, RPS2 transcript levels in the fec and bbc mutant plants were similar to those in Col-0 plants after inoculation of indicated bacterial strains. Statistical analysis was performed using two-way ANOVA with Tukey’s test. (mean ± s.e.m.; n = 3 biologically independent samples). c, Phosphorylation of MPK3 and MPK6 (MPK3/6) in Col-0 and the PRR/co-receptor mutants after D36E or D36E(avrRpt2) inoculation. An equal amount of total protein was loaded in each lane. Experiments were repeated at least three times with similar trends. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 3 Characterization of different lines of bbc/Dex:avrRpt2 plants.
a, Schematic of the experimental design. Leaf discs were first treated with flg22 + dexamethasone (Dex) for 35 min, and the production of ROS was detected by a microplate reader. Leaf discs were then washed with sterilized water 4 times, for 5 min each time. Sterilized water (mock treatment), 100 nM flg22, 5 μM dexamethasone or 100 nM flg22 + 5 μM dexamethasone was then added for detection of second-phase ROS. b, Expression levels of the avrRpt2 transgene in different transgenic lines 2 h after infiltration with 5 μM dexamethasone. Statistical analysis was performed using one-way ANOVA with Tukey’s test (mean ± s.e.m.; n = 3 (Col-0/Dex:avrRpt2 L2, bbc/Dex:avrRpt2 L1, bbc/Dex:avrRpt2 L2) or 4 (Col-0/Dex:avrRpt2 L1) biologically independent samples). Experiments were repeated three times with similar trends.
Extended Data Fig. 4 AvrRpt2-triggered ETIROS depends on NADPH oxidase.
a–c, ROS production in Col-0/Dex:avrRpt2 L1 plants was inhibited by NADPH oxidase inhibitor DPI. Leaf discs were treated with 100 nM flg22 and 5 μM dexamethasone (Dex). DPI, SHAM and NaN3 were added at the beginning of measurement (mean ± s.e.m.; n (numbers of leaf discs) are indicated in the panel). b, c, Total photon counts are calculated from a at the PTI phase (0–30 min) or ETI phase (60–200 min). Statistical analysis was performed by one-way ANOVA with Tukey’s test. d, ETI-associated ROS burst is inhibited by DPI, an NADPH oxidase inhibitor. ROS was detected in Col-0/Dex:avrRpt2 plants after treatment with 100 nM flg22 and 5 μM dexamethasone. Chemical inhibitors (DPI, SHAM or NaN3) were added after the first ROS burst (about 40 min after addition of flg22 and dexamethasone). Data are mean ± s.e.m. n (numbers of leaf discs) is indicated in the panel. In box plots the centre line represents the median, box edges delimit lower and upper quartiles and whiskers show the highest and lowest data points. Experiments in this figure were repeated three times with similar trends.
Extended Data Fig. 5 The rbohd and bik1 mutant plants are compromised in ETI resistance against Pst DC3000(avrRpt2).
a, Appearance of the 5 week-old rbohd mutant plants before bacteria inoculation. b, Disease symptom of Col-0 and rbohd mutant plant 2 days after Pst DC3000 and Pst DC3000 (avrRpt2) infiltration. c, Appearance of the 4.5 week-old bik1 mutant plants growth in Redi-Earth soil before bacteria inoculation. d, Disease symptoms of Col-0 and bik1 mutant plant 2 days after Pst DC3000 and Pst DC3000 (avrRpt2) infiltration. Experiments in this figure were repeated three times with similar trends.
Extended Data Fig. 6 The AvrRpt2 ETI-associated ROS burst is partially mediated by BIK1.
a, ROS was detected in the bik1 and cpk5 cpk6 cpk11 mutant plants by H2DCFDA dye 4.5 h after D36E(avrRpt2) inoculation. Scale bars, 25 μm. b, ROS was detected in the bik1 mutant plants by H2DCFDA dye 5 h after D36E or D36E(avrRpt2) inoculation. Plants were grown on 0.5× Murashige–Skoog plates for 3 weeks. Scale bars, 25 μm. Experiments in this figure were repeated three times with similar trends.
Extended Data Fig. 7 Transcriptomic analysis of RNA-seq experiments.
a, A diagram showing the RNA-seq design in this study. b, Bacterial population in Arabidopsis leaves at 3 h or 6 h post infiltration. Data are mean ± s.d. (n = 3 biologically independent samples). Statistical analysis was performed using two-way ANOVA with Tukey’s test. c, A Venn diagram showing numbers of differentially expressed genes (DEGs) 3 h after D36E or D36E(avrRpt2) infection in Col-0 plants. d, Heat map of the expression pattern of D36E/PTI-responsive genes. e–g, Heat maps of salicylic acid-responsive (e; genes extracted from ref. 42), jasmonate-responsive (f; genes extracted from ref. 43) and ethylene-responsive (g; genes extracted from ref. 44) genes.
Extended Data Fig. 8 PRR/co-receptors are important for immune-related gene expression.
a, b, WRKY–FRK1 is a unique immune branch and cannot be restored by ETI in the bbc mutant. a, Heat map of the 272 DEGs in the bbc plant compared to Col-0 plant after D36E(avrRpt2) infection, with the canonical PTI pathway genes highlighted in red. b, RT–qPCR of FRK1 and WRKY29 expression level in Col-0 and bbc plants 3 h after infiltration with different strains or mock treatment (mean ± s.e.m.; n = 3 biologically independent samples; statistical analysis by two-way ANOVA with Tukey’s test). c, Expression level of avrRpt2, AZI1, EARLI1 and AZI4 in the Col-0/Dex:avrRpt2 L1 and bbc/Dex:avrRpt2 L2 plants after sterilized water (mock) or dexamethasone (Dex, 50 nM for Col-0/Dex:avrRpt2 and 100 nM for bbc/Dex:avrRpt2) treatment. Leaves were collected 2 h post-infiltration for transcript analysis (mean ± s.e.m.; n = 3 biologically independent samples; statistical analysis by two-way ANOVA with Tukey’s test). Experiments in b and c were repeated at least three times with similar trends.
Extended Data Fig. 9 Heat map of RLK–LYK5–RLP-pathway and BIK1/PBL family gene expression.
a, RLK–LYK5–RLP-pathway gene expression by RNA-seq. b, BIK1/PBL family gene expression by RNA-seq. Numerical values indicate expression level calculated as TPM. Genes labelled in red show significant upregulation after D36E(avrRpt2) inoculation, compared to mock and D36E inoculation, in Col-0 and bbc plants. Arrows in b indicate BIK1 and PBL1 genes.
Extended Data Fig. 10 Upregulation of key PTI component genes by AvrRpt2-triggered ETI seems to be independent of PTI and salicylic acid–N-hydroxy-pipecolic acid.
a, RT–qPCR results of representative PTI-pathway genes. Col-0 and bbc plants were infiltrated with different strains indicated, and leaves were collected 3 h post infiltration for transcript analysis (mean ± s.e.m.; n = 3 biological replicates for all plants or genes; except bbc-BAK1, n = 4 biologically independent samples). Statistical analysis by two-way ANOVA with Tukey’s test. P values for additional comparisons are provided in Supplementary Table 3. b, RT–qPCR analysis of BIK1, XLG2, MKK4, MKK5 and MPK3 expression levels in Col-0 and sid2 plants 3 h after infiltration with D36E or D36E(avrRpt2). Statistical analysis by two-way ANOVA with Tukey’s test (mean ± s.e.m.; n = 3 (for Col-0) or 4 (for sid2) biologically independent samples). These experiments were repeated at least three times with similar trends. c, Heat maps of N-hydroxy-pipecolic acid-responsive genes (extracted from ref. 45, defined by genes that are responsive to pipecolic acid and depend on FMO1 for expression) in the Col-0 and bbc plants in our RNA-seq experiment.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1: Uncropped images of protein blots; Supplementary Table 2: Primers used in this study; and Supplementary Table 3: P-values of additional comparisons in Figure 2d, 4a and Extended Figure 10a.
Supplementary Table 1
Differentially expressed genes in Col-0 and bbc, as determined by DESeq R packag and using a negative binomial distribution-based model. P-values were adjusted using the Benjamini and Hochberg’s approach and genes with an padj-value < 0.05 and log2(Fold change) > 1 are shown.
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Yuan, M., Jiang, Z., Bi, G. et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 592, 105–109 (2021). https://doi.org/10.1038/s41586-021-03316-6
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DOI: https://doi.org/10.1038/s41586-021-03316-6
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