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Quinone perception in plants via leucine-rich-repeat receptor-like kinases

Nature volume 587, pages 92–97 (2020)Cite this article

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Abstract

Quinones are produced and sensed in all kingdoms of life1,2,3,4. Plants are primary producers of quinone1,2, but the role of quinone as a signalling agent in plants remains largely unknown. One well-documented role of quinone is in the induction of haustoria (specialized feeding structures) in plants that parasitize roots, which occurs in the presence of the host-derived quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ)5. However, how parasitic plants sense DMBQ remains unclear, as is whether nonparasitic plants are capable of sensing quinones. Here we use Arabidopsis thaliana and DMBQ as a model plant and quinone to show that DMBQ signalling occurs in Arabidopsis via elevation of cytosolic Ca2+ concentration. We performed a forward genetic screen in Arabidopsis that isolated DMBQ-unresponsive mutants, which we named cannot respond to DMBQ 1 (card1). The CANNOT RESPOND TO DMBQ 1 (CARD1; At5g49760, also known as HPCA1) gene encodes a leucine-rich-repeat receptor-like kinase that is highly conserved in land plants. In Arabidopsis, DMBQ triggers defence-related gene expression, and card1 mutants show impaired immunity against bacterial pathogens. In Phtheirospermum japonicum (a plant that parasitizes roots), DMBQ initiates Ca2+ signalling in the root and is important for the development of the haustorium. Furthermore, CARD1 homologues from this parasitic plant complement DMBQ-induced elevation of cytosolic Ca2+ concentration in the card1 mutant. Our results demonstrate that plants—unlike animals and bacteria—use leucine-rich-repeat receptor-like kinases for quinone signalling. This work provides insights into the role of quinone signalling and CARD1 functions in plants that help us to better understand the signalling pathways used during the formation of the haustorium in parasitic plants and in plant immunity in nonparasitic plants.

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Fig. 1: DMBQ induces several hallmarks of a signalling event in Arabidopsis, and is sensed by CARD1.
Fig. 2: Cysteine residues 395 and 405 are important for DICE mediated by CARD1.
Fig. 3: CARD1 is required for resistance to bacterial infection.
Fig. 4: CARD1 homologues in Phtheirospermum japonicum contribute to DMBQ signalling.

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Data availability

All data in this study are available in the Article and its Supplementary Information. Accession numbers of sequences used in this study are given in Supplementary Table 3. RNA-seq data from this study have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE128510. All other data, such as raw tree files and raw chromatography trace files due to the size limitation, are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank M. Krebs, M. Hayashi and J. M. Davies for sharing materials; M. Seo for sharing the thermal imaging system; T. Umehara and N. Tochio for training and access to the dynamic light scattering instrument; S. Morino, A. Shibata, H. Hanasaki, T. Shinagawa and A. Furuta for help in the laboratory; T. Spallek for providing Striga asiatica gDNA and comments on the manuscript; P. Schulze-Lefert, J. Mortimer and P. Gan for comments on the manuscript; Y. Kadota for discussions; and assistants at the laboratory of K.S. for support. This work was partially supported by RIKEN Special Postdoctoral Researcher program to A.L. and by Ministry of Education, Culture, Sports, Science and Technology KAKENHI grants (15H05959 and 17H06172 to K.S. and 15H05955 to T.S.).

Author information

Author notes

  1. These authors contributed equally: Anuphon Laohavisit, Takanori Wakatake.

Authors and Affiliations

  1. RIKEN Center for Sustainable Resource Science, Yokohama, Japan

    Anuphon Laohavisit, Takanori Wakatake, Nobuaki Ishihama, Hugh Mulvey, Kaori Takizawa & Ken Shirasu

  2. Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Takanori Wakatake & Ken Shirasu

  3. College of Bioscience and Biotechnology, Chubu University, Kasugai, Japan

    Takamasa Suzuki

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  1. Anuphon Laohavisit
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Contributions

A.L. and K.S. conceived, designed and coordinated the project. A.L. performed initial characterization of DMBQ signalling, generated the ethylmethanesulfonate-mutagenized population, performed genetic screens, validated genetic interactions and created the mapping population. T.S. performed mutant sequencing and mapping. A.L., with assistance from H.M. and K.T., performed cloning, transformation, DNA library constructions, western blotting experiments and further characterization of DMBQ signalling in A. thaliana. A.L. performed RNA-seq analyses, and the pathogen and stomatal assays. T.W. performed R-GECO1 experiments, generated constructs for experiments in P. japonicum, performed genetic manipulation in P. japonicum and characterized DMBQ signalling in P. japonicum. N.I. designed and purified CARD1 antibodies, expressed and purified ectoCARD1 protein and performed the binding assay. A.L., T.W. and N.I. wrote the original draft. A.L., H.M. and K.S. reviewed and edited with input from all of the authors.

Corresponding author

Correspondence to Ken Shirasu.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of DMBQ-induced [Ca2+]cyt elevation in Arabidopsis.

a–c, DICE showed different spatiotemporal dynamics to flg22-induced [Ca2+]cyt elevation in Arabidopsis. Kymographs of R-GECO1 signal in Arabidopsis in response to 10 μM DMBQ (a), 1 μM flg22 (b) or mock (c). Kymographs were extracted from time-lapse images (Supplementary Videos 1–3) along three-pixel lines as shown on the left panels. For b, two kymographs were obtained from two types of root tissues (epidermis and vasculature), which are shown in white and yellow lines, respectively. Stimuli were added at 0 min. Signal intensity is shown in rainbow spectrum. Scale bar, 100 μm. d, DMBQ does not induce apoplastic ROS production. External ROS production was measured against DMSO (control), DMBQ or flg22 (positive control), using a chemiluminescence probe (L-012). Data are mean ± s.d. from 48 seedlings. The experiment was repeated twice, with similar results obtained. e, DMBQ induces [Ca2+]cyt elevation in Arabidopsis seedlings in a dose-dependent manner, following Michaelis–Menten kinetics. f, Maximum concentration of Ca2+ influx at any given DMBQ concentration using data from e. g, Lineweaver–Burk plot of f. Km was calculated from x-axis intercept. Pretreatment with protein kinase inhibitor or chemicals that interfere with Ca2+ influx reduced DICE in a dose-dependent manner. h–j, WTAEQ Arabidopsis seedlings were pretreated with K252a (n = 6 seedlings) (h), LaCl3 (n = 4 seedlings) (i) or EDTA (n = 6 seedlings) (j) for 30 min before addition of 5 μM DMBQ concentration, as indicated. Data are mean ± s.d. Error bars are indicated every 30 s for clarity. For a–c, e–g, experiments were repeated 3 times with similar results obtained.

Source data

Extended Data Fig. 2 Forward genetic screen isolates DMBQ non-responsive mutants.

a, M2 seedlings were grown in vitro in a white 96-well plate, and used to screen against DMBQ. An image of the seedlings is shown on the left and the results obtained after the seedlings were challenged with DMBQ (5 μM) are shown on the right. Red circles indicate wells that did not have any seedlings, or in which seedlings are very small and give no signals. Yellow circles indicate wells that contained viable seedlings and showed a reduction in DMBQ response. Blue circles indicate seedlings that were viable but were not responsive to DMBQ. These plants were rescued and transferred onto the soil. b, Strategy to further narrow down mutant candidates. c, Results of the homozygosity tests and replications for each candidate. d, eATP response is normal in these candidates. For c and d, experiments were repeated three times with similar results. e, BC1F1 population from each candidate showed semi-dominant phenotypes. f, Allelism tests suggest that these candidates most probably have the mutation in the same gene. For e and f, experiments were performed twice with similar results. g, Table indicating which lines were crossed to perform allelism tests. Line 8 and line 10 were not tested as they were late-flowering compared to the other lines. h, Diagram of CARD1 gene structure, showing exons in blue boxes and introns as lines in between them. Point mutation positions in different card1 alleles, and T-DNA insert positions, are indicated. i, CARD1 is predominantly localized to the plasma-membrane-enriched fraction. Plasma membrane (PM), organelle membrane (OM) and soluble fraction (SF) were separated, and each fraction was probed with anti-CARD1, anti-AHA1 (plasma membrane marker), anti SMT1 (endoplasmic reticulum membrane marker), and anti-RbcL (soluble fraction marker). The localization of CARD1 in card1 loss-of functions alleles, which retained some protein expression, was also assessed. Experiments were performed twice with similar results.

Source data

Extended Data Fig. 3 Structure–activity relationship between CARD1-dependent [Ca2+]cyt elevation and various chemicals.

Various quinone-related compounds (red, teal, orange and purple dashed boxes) are capable of inducing [Ca2+]cyt elevation in Arabidopsis seedlings in a CARD1-dependent manner. Redox-related compounds (green dashed box) induce [Ca2+]cyt elevation in Arabidopsis seedlings, but are CARD1-independent, whereas lignin-related compounds (brown dashed box) cannot induce [Ca2+]cyt elevation at mid-micromolar concentration. CARD1 is not involved in sensing redox-related molecules such as reduced glutathione, nitic oxide generated via S-nitroso-N-acetyl-d,l-penecillamine (SNAP) and N-acetylcysteine. Possible products of lignin breakdown (acetosyringone and sinapic acid) do not induce [Ca2+]cyt elevation, similar to the responses for sinapyl and conifyl alcohols in Fig. 1b. Compounds (5 μM, unless otherwise indicated) were tested for their ability to induce [Ca2+]cyt elevation in WTAEQ and card1-2 Arabidopsis seedlings. Compounds are grouped according to their structures. Red arrows indicate the timing for stimuli addition. Data are mean ± s.d., n = 12 seedlings per genotype per chemical.

Source data

Extended Data Fig. 4 Some quinones can induce [Ca2+]cyt elevation at low-nanomolar concentrations.

Chemical structure for each quinone is shown in the left-hand column. Dose-dependent analysis was carried out as in Extended Data Fig. 1. For each chemical, the maximum concentration of Ca2+ influx at any given quinone concentration was plotted and the graph was transformed to obtain a Lineweaver–Burk plot. Km values were calculated and are shown below each chemical structure. Experiments were repeated twice with similar results.

Source data

Extended Data Fig. 5 Conservation of CARD1 homologues in land plants.

CARD1 homologues are present throughout land plant lineages, but not in chlorophytes, and can be separated into two distinct groups. a, A cladogram representing the phylogeny of different Viridiplantae species on the basis of the Phytozome tree representation (database version 12.1). Bars indicate the number of CARD1 homologues in each species. Specific clades are denoted in red. b, A phylogenetic tree of CARD1 and its homologues in Marchantia polymorpha (Mapoly), Physcomitrella patens (Pp), Oryza sativa (Os), Brachypodium distachyon (Bradi), Phtheirospermum japonicum (Pj) Striga asiatica (Sa) and Arabidopsis thaliana (At) was mined from the Phytozome version 12.1 database, and full protein sequences were aligned using MUSCLE (default parameters). A maximum-likelihood tree was generated by RAxML and rooted with Mapoly0097s0050. Arabidopsis CARD1 is denoted in red. Numbers on branches indicate bootstrap support values out of 200 bootstrap replicates. c, A unique cysteine region is highly conserved between CARD1 and its homologues across different species in Viridiplantae. A partial sequence alignment of CARD1 and its homologues used to create the phylogenetic tree in b. The unique cysteine region is highlighted in yellow. d, A unique cysteine region is conserved within LRR-RLK subfamily VIII-1, but not in other groups within the LRR-RLKs. A partial sequence alignment of CARD1 and its homologues (LRR-RLK subfamily VIII-1) with other LRR-RLKs. Representatives from each LRR-RLK group (marked within parentheses after gene name) were used to perform alignment with MUSCLE using default parameters. The unique cysteine region is boxed in yellow. Conserved cysteine residues are annotated (relative to Arabidopsis CARD1 numbering) and marked with asterisks. Arabidopsis CARD1 is denoted in red. Accession numbers are given in Supplementary Table 3.

Extended Data Fig. 6 DMBQ substantially induces CARD1-dependent defence- and stress-related transcripts.

a, Confirmation of several DMBQ induced stress-related transcripts at 10 min by qPCR. Relative fold difference upon DMBQ treatment for various stress-related genes (n = 3 samples, 20 seedlings per sample). Comparison between fold difference values are shown on the right as obtained from qPCR (top row) or RNA-seq data (bottom row). b, Relative changes in gene transcripts for WTAEQ and card1-2 plants upon DMBQ treatment at 30 min, 1 h and 6 h. Volcano plots representing global changes in gene transcript expression upon DMBQ treatment for WTAEQ and card1-2 plants at 30 min, 1 h and 6 h, as indicated. Red dots denote gene transcripts that showed log2-transformed fold change >1 and FDR <0.05. Blue denotes transcripts that showed log2-transformed fold change <1 and FDR <0.05. Green denotes transcripts that showed log2-transformed fold change >1 and FDR >0.05. The number of transcripts that showed log2-transformed fold change >1 and FDR <0.05 in WTAEQ and card1-2 plants was used to plot Venn diagrams for each time point. Only genes that were differentially regulated in WTAEQ plants (quasi-likelihood F-test) were used to perform Gene Ontology analysis. Gene Ontology analysis P values (Fisher’s exact test with Bonferroni corrections) are shown adjacent. n = 3 samples per genotype per treatment (20 seedlings per sample). c, The surface temperature of leaves in card1 plants is cooler than the complemented line upon DMBQ treatment. Thermal images of soil-grown five-week-old card1-2 plants and the complemented line (card1-2/pCARD1::CARD1) 3 h after plants were challenged with DMBQ (50 μM). Experiments were performed twice with similar data obtained. d, DMBQ pretreatment did not increase resistance to Pst DC3000 infection. The complemented line and card1-2 plants were sprayed with DMSO control or 50 μM DMBQ and left in the growth chamber for 3 h before inoculation with Pst DC3000 (spray inoculation, OD600 = 0.02). Colony-forming units per cm2 were determined 3 days post-inoculation. No significantly different means at P < 0.05 were observed (two-sided t-test; n = 6 biologically independent samples). The experiment was repeated twice with similar results. For a, d, box plot elements are as follows; centre lines indicate the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles.

Source data

Extended Data Fig. 7 DMBQ-induced [Ca2+]cyt elevation in P. japonicum is important for haustorium formation.

a–c, DICE in P. japonicum is confined to, and correlated, with the future site of haustorium emergence. Kymographs of R-GECO1 signal in P. janonicum hairy roots in response to 10 μM DMBQ (a), 1 μM flg22 (b) or mock (c). Kymographs were extracted from time-lapse images (Supplementary Videos 4–6) along three-pixel lines as shown on the left panels. Stimuli were added at 0 min. Signal intensity is shown in rainbow spectrum. Scale bar, 100 μm. Experiments were repeated three times with similar results. d, La3+ and K252a inhibit DMBQ-induced haustorium formation in a dose-dependent manner. Above 10 μM, La3+ showed toxicity to P. japonicum roots, whereas up to 5 μM K252a did not show any toxic effect. Images on the right are magnified from the left. Red asterisks indicate haustorium formed in root tips. Scale bar, 1 mm. e, Quantitative analysis of data from d for the La3+ effect at indicated concentrations. Haustorium formation was observed under a dissecting microscope and the frequency was scored (n = 3; 10 seedlings per n). The experiment was repeated three times with similar results. Different letters indicate significantly different means at P < 0.05 (one-way ANOVA, Tukey HSD post hoc test). f, As in e, but for K252a (n = 3; 10 seedlings per n). The experiment was repeated three times with similar results. Different letters indicate significantly different means at P < 0.05 (one-way ANOVA, Tukey HSD post hoc test). Images are in d. For e, f, box plot elements are as follows; centre lines indicate the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. g, Haustorium formation is induced at least after 30 min of DMBQ treatment and can be inhibited by La3+ or K252a. Representative images of different treatments for Fig. 4d, e. Images on the right are magnified from the left. Red asterisks indicate haustorium formed in root tips. Scale bar, 1 mm.

Source data

Extended Data Fig. 8 Structure–activity relationship between PjCADL-dependent [Ca2+]cyt elevation, haustorium formation and various chemicals.

Various quinone-related compounds can induce [Ca2+]cyt elevation. a, Top, DMSO control. Bottom, 10 μM DMBQ. b–q,Top panels, calcium traces in response to different chemicals. Compounds (10 μM, unless otherwise indicated) were tested for their ability to induce [Ca2+]cyt elevation in card1-2 seedlings complemented with CARD1 (blue), empty control (orange), PjCADL1 (grey), PjCADL2 (yellow) and PjCADL3 (dark blue). Data are mean ± s.d., n = 12 seedlings per genotype per chemical. Bottom panels, images of haustorium formed in P. japonicum seedlings after corresponding chemical treatment. Images on the right are magnified from the left. Red asterisks indicate haustorium formed in root tips. Scale bar, 1 mm. r, Haustorium formation scored from a to q (n = 3 samples; 10 seedlings per sample). Box plot elements are as follows; centre lines indicate the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. The experiment was repeated 3 times with similar results.

Source data

Extended Data Fig. 9 RNA interference experiment of PjCADL genes.

a, Gene models of PjCADL genes. Positions of RNA interference targets and qPCR primers are indicated. b, Expression level of PjCADL genes in Arabidopsis card1-2/pCARD1::PjCADL1, card1-2/pCARD1::PjCADL12 and card1-2/pCARD1::PjCADL13, as determined by qPCR. n = 3 samples (20 seedlings per sample). c, Schematic of P. japonicum transformation and RNA interference experiment. d, Representative images of different haustorium phenotypes observed from hairy-root transformants from RNA interference experiment. Scale bar, 100 μm. e, Summary of different sample pools obtained from P. japonicum RNA interference experiment. f, Transcript levels of PjCADL genes relative to PjUBC2 in each pool samples from e. n = 3 independent trials. Each trial used the same RNA from pool samples from e, but new reverse transcription and qPCR experiments were performed for each trial. Box plot elements are as follows; centre lines indicate the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. The experiment was repeated three times with similar results.

Source data

Extended Data Fig. 10 Interaction between CARD1 ectodomain and DMBQ in vitro.

a, Strategic overview of ectoCARD1 protein purification and binding assay. b, A fast protein liquid chromatography trace of extracted apoplastic fluid subjected to anion exchange column with 1 M NaCl gradient. Inset shows a magnified trace, and fractions collected. Each fraction (A to M) was subjected to SDS–PAGE to determine fractions with ectoCARD1. A′, apoplastic fluid. c, Fractions D–G were pooled and subjected to size exclusion chromatography. Inset represents a magnified trace and fractions collected. Each fraction (O to W) was subjected to SDS–PAGE to determine the presence of ectoCARD1. B′, pooled D–G fraction. For b, c, experiments were repeated three times with similar data obtained. d, Purified ectoCARD1 behaves as a monomer and did not form protein aggregations. Purified ectoCARD1 was subjected to a dynamic light scattering (DLS) experiment. Size distribution was determined by intensity. Data are from three different scans on a protein sample, with mean Z-average and polydispersity index indicated. Experiment was repeated three times with similar results. Predicted and experimentally determined ectoCARD1 protein sizes are indicated below. Molecular size calculation from analytical size exclusion chromatography (SEC) was determined from protein standards. Molecular size calculation from DLS instrument was determined using Zetasizer software. The slightly higher molecular size calculated from analytical size exclusion chromatography and DLS than predicted may indicate possible glycosylation and other post-translational modification of ectoCARD1. e, In vitro binding assay between DMBQ and ectoCARD1. Isothermal titration calorimetry experiment was performed by titrating DMBQ into ectoCARD1. Top, raw trace of isothermal titration calorimetry experiment. Bottom, enthalpy change determined from the raw trace. Experiment was repeated three times with similar results.

Supplementary information

Supplementary Figure

Supplementary Figure. Uncropped blots and stained membranes.

Reporting Summary

Supplementary Data

Supplementary Data. List of CARD1-dependent genes from RNA-seq analysis in response to 5 µM DMBQ for 10 min, 30 min, 1 hr and 6 hr. WTAEQ and card1-2 seedlings were treated with DMBQ treatment and genes which were differentially regulated in WTAEQ (Log2FC > 1 and FDR < 0.05; quasi-likelihood F-test) are listed. There were no CARD1-dependent transcript which were downregulated (Log2FC > 1 and FDR < 0.05; quasi-likelihood F-test) at 10 min after treatment. n = 4 samples for 10 min and n = 3 samples for 30 min, 1 hr and 6 hr, per genotype per treatment (20 seedlings per sample).

Supplementary Tables

Supplementary Table 1. List of chemicals used in this study which were not purchased from Fujifilm Wako. Supplementary Table 2. List of primers used in this study. Supplementary Table 3. Accession numbers of sequences used in this study.

Video 1

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing Arabidopsis seedlings in response to 10 µM DMBQ. Root of 5-day old A. thaliana expressing R-GECO1 was imaged upon challenged with 10 µM DMBQ. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results. Scale bar, 50 µm.

Video 2

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing Arabidopsis seedlings in response to 1 µM flg22. Root of 5-day old A. thaliana expressing R-GECO1 was imaged upon challenged with 1 µM flg22. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results. Scale bar, 50 µm.

Video 3

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing Arabidopsis seedlings in response to DMSO control. Root of 5-day old A. thaliana expressing R-GECO1 was imaged upon challenged with DMSO control. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results.Scale bar, 50 µm.

Video 4

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing P. japonicum hairy root in response to 10 µM DMBQ. Freshly elongated P. japonicum with R-GECO1-transformed hairy roots was imaged upon challenged with 10 µM DMBQ. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results. Scale bar, 100 µm.

Video 5

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing P. japonicum hairy root in response to 1 µM flg22. Freshly elongated P. japonicum with R-GECO1-transformed hairy roots was imaged upon challenged with 1 µM flg22. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results. Scale bar, 100 µm.

Video 6

: [Ca2+]cyt dynamics in the root of R-GECO1-expressing P. japonicum hairy root in response to DMSO. Freshly elongated P. japonicum with R-GECO1-transformed hairy roots was imaged upon challenged with DMSO control. Images were taken for 11 minutes at intervals of 2 seconds. Stimulus was applied 1 minute after starting measurement. Experiments were repeated 3 times with similar results. Scale bar, 100 µm.

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Laohavisit, A., Wakatake, T., Ishihama, N. et al. Quinone perception in plants via leucine-rich-repeat receptor-like kinases. Nature 587, 92–97 (2020). https://doi.org/10.1038/s41586-020-2655-4

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