Perception of a conserved family of plant signalling peptides by the receptor kinase HSL3
- The Sainsbury Laboratory, University of East Anglia, United Kingdom
- The Plant Signaling Mechanisms Laboratory, Department of Plant Molecular Biology, University of Lausanne, Switzerland
- Institute of Plant Molecular Biology, Zurich‐Basel Plant Science Center, University of Zurich, Switzerland
- MWSchmid GmbH, Switzerland
Figures
Figure 1 with 2 supplements
CTNIPs are a novel family of plant signalling peptide.
(a) Heat map showing log2(FC) expression levels of CTNIP1–4 in response to a range of elicitors (data obtained from Bjornson et al., 2021). CTNIP5 was excluded as it is unannotated in the TAIR10 …
-
Figure 1—source data 1
CTNIPs are a novel family of plant signalling peptide.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig1-data1-v2.xlsx
-
Figure 1—source data 2
CTNIP-induced MAPK phosphorylation.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig1-data2-v2.pdf
Figure 1—figure supplement 1
Alignment and phylogeny of Arabidopsis CTNIPs.
(a) Expression heat map showing log2(FC) expression levels of CTNIP1, CTNIP2, CTNIP3, and CTNIP5 taken from ArrayExpress which mapped to the Araport11 annotation (Athar et al., 2019; Bjornson et …
-
Figure 1—figure supplement 1—source data 1
Expression levels of CTNIP1, CTNIP2, CTNIP3, and CTNIP5.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Characterisation of CTNIP synthetic peptides.
(a) Alignment of CTNIP4 peptide sequences used in this manuscript. (b) Western blot using α-p42/p44-ERK recognising phosphorylated MAP kinases in seedlings treated with 100 nM CTNIP4 fragments or …
-
Figure 1—figure supplement 2—source data 1
CTNIP-induced calcium influx.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig1-figsupp2-data1-v2.xlsx
-
Figure 1—figure supplement 2—source data 2
CTNIP-induced MAPK phosophorylation.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig1-figsupp2-data2-v2.pdf
Figure 2 with 3 supplements
HAESA-LIKE 3 (HSL3) forms a CTNIP-induced receptor complex with BAK1.
(a) Schematic representation of BAK1-GFP immunoprecipitation in the (1) absence or (2) presence of CTNIP4 treatment to identify protein associations induced by CTNIP. Figure generated using …
-
Figure 2—source data 1
HSL3-specific spectral counts.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig2-data1-v2.xlsx
-
Figure 2—source data 2
Westernblots showing affinity purification of BAK1 with HSL3-GFP.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig2-data2-v2.pdf
Figure 2—figure supplement 1
Phylogeny of Arabidopsis leucine-rich repeat receptor kinase (LRR-RK) subfamily XI.
Phylogeny of full-length protein sequences of the Arabidopsis LRR-RK subfamily XI. Sequences were aligned using MUSCLE and a phylogeny was inferred using the maximum-likelihood method and JTT …
-
Figure 2—figure supplement 1—source data 1
Flg22-induced expression data of LRR-RK subfamily XI.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Isothermal titration calorimetry (ITC) independent experiments and purification of HAESA-LIKE 3 (HSL3) and BAK1 used in the binding experiments.
(a) Analytical size exclusion chromatography (SEC) of the ectodomains of HSL3 and BAK1. An SDS-PAGE of the two proteins is shown alongside. (b) ITC raw thermograms of experiments shown in the ITC …
Figure 2—figure supplement 3
Structural comparison of the binding pockets between the receptors HAESA and HAESA-LIKE 3 (HSL3).
(a) The hydroxyproline pocket required for anchoring the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide to the HAESA receptor is missing in HSL3. Close view of the binding pocket of the …
Figure 3 with 5 supplements
HAESA-LIKE 3 (HSL3) is strictly required for CTNIP perception and growth regulation.
(a) Western blot using α-p42/p44-ERK recognising phosphorylated MAP kinases in seedlings treated with 100 nM CTNIPs or mock for 15 min. The membrane was stained with Coomassie brilliant blue (CBB), …
-
Figure 3—source data 1
HSL3 is strictly required for CTNIP perception and growth regulation.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-data1-v2.xlsx
-
Figure 3—source data 2
HSL3-dependency of CTNIP-induced MAPK phosphorylation.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-data2-v2.pdf
Figure 3—figure supplement 1
Genetic characterisation of hsl3 mutants.
(a) Gene model showing the location of T-DNA inserts. (b) PCR confirming T-DNA insertion and mutant homozygosity.
-
Figure 3—figure supplement 1—source data 1
Genetic characterisation of hsl3 mutants.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-figsupp1-data1-v2.pdf
Figure 3—figure supplement 2
CTNIP-induced seedling growth inhibition.
(a–b) Fresh weight of 14-day-old seedlings grown in the presence of 500 nM CTNIPs for 10 days relative to mock (n = 8 seedlings). A line represents mean; error bars represent SD. p-Values indicate …
-
Figure 3—figure supplement 2—source data 1
CTNIP-induced seedling growth inhibition.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
Correlation of CTNIP4-induced transcriptomic response with that of elicitors at 30min.
CTINP4-induced gene expression is well correlated with elicitor-induced gene expression from Bjornson et al., 2021. Circle colour and size are proportional to the Spearman correlation coefficient …
-
Figure 3—figure supplement 3—source data 1
Correlation of CTNIP4-induced transcriptomic response with that of elicitors at 30min.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-figsupp3-data1-v2.csv
Figure 3—figure supplement 4
Characterisation of CTNIP and chimeric receptor lines.
(a) Western blot using α-FLAG recognising BIR3ecto-HSL3cyto-FLAG in seedlings to confirm expression. The membrane was stained with Coomassie brilliant blue (CBB), as a loading control. (b) …
-
Figure 3—figure supplement 4—source data 1
Characterisation of CTNIP and chimeric receptor lines.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-figsupp4-data1-v2.pdf
Figure 3—figure supplement 5
HSL3-CTNIP mutants do not show altered resistance to Pseudomonas syringae pv tomato DC3000 ΔAvrPto/ΔAvrPtoB.
Four-week-old plants were spray inoculated with P. syringae pv tomato DC3000 ΔAvrPto/ΔAvrPtoB (OD600=0.2). Data points represent individual plants (n=8). Red bars represent medians. Dashed lines …
-
Figure 3—figure supplement 5—source data 1
HSL3-CTNIP mutants do not show altered resistance to Pseudomonas syringae pv tomato DC3000 ΔAvrPto/ΔAvrPtoB.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig3-figsupp5-data1-v2.xlsx
Figure 4 with 1 supplement
The HSL3-CTNIP signalling module predates extant angiosperms.
(a) Phylogeny of the full-length amino acid sequences of HAE/HSL/CEPR/RLK7/IKU2 clade of receptor kinases. Eudicot sequences are indicated in blue, monocot sequences in green, and Amborella …
-
Figure 4—source data 1
The HSL3-CTNIP signalling module predates extant angiosperms.
- https://cdn.elifesciences.org/articles/74687/elife-74687-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
HAE/HSL/CEPR/RLK7/IKU2 clade phylogenies.
Phylogenies of HAE/HSL/CEPR/RLK7/IKU2 clade LRR domains and kinase domains. Eudicot sequences are indicated in blue, monocot sequences in green, and Amborella sequences in red. Corresponding …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | HSL3 | ARAPORT11 | AT5G25930, Q9XGZ2_ARATH | |
| Gene (Arabidopsis thaliana) | CTNIP1 | ARAPORT11 | AT1G06135, Q8LCX3_ARATH | |
| Gene (Arabidopsis thaliana) | CTNIP2 | ARAPORT11 | AT1G06137, F4IBZ9_ARATH | |
| Gene (Arabidopsis thaliana) | CTNIP3 | ARAPORT11 | AT2G31335, Q1G3B9_ARATH | |
| Gene (Arabidopsis thaliana) | CTNIP4 | ARAPORT11 | AT2G31345, Q8L9Z1_ARATH | |
| Gene (Arabidopsis thaliana) | CTNIP5 | ARAPORT11 | AT2G23123, A0A1I9LM80_ARATH | |
| Gene (Medicago truncatula) | MtHSL3 | Mt4.0 | G7J3I8_MEDTR, MTR_3g110450, MtrunA17_Chr3g0140551 | |
| Gene (Medicago truncatula) | MtCTNIP | Mt4.0 | G7I613_MEDTR, MTR_1g044470, MtrunA17_Chr1g0168241 | |
| Genetic reagent (Arabidopsis thaliana) | hsl3-1 | euNASC | salk_207895 | |
| Genetic reagent (Arabidopsis thaliana) | hsl3-2 | euNASC | wiscdslox450b04 | |
| Genetic reagent (Arabidopsis thaliana) | bak1-4/pBAK1::BAK1-GFP | https://doi.org/10.1105/tpc.111.090779 | bak1-4/pBAK1::BAK1-GFP | |
| Genetic reagent (Arabidopsis thaliana) | bak1-5 | https://doi.org/10.1371/journal.pgen.1002046 | bak1-5 | |
| Genetic reagent (Arabidopsis thaliana) | p35S::AEQUORIN | https://doi.org/10.1038/352524a0 | ||
| Genetic reagent (Nicotiana benthamiana) | p35S::AEQUORIN | https://doi.org/10.1104/pp.110.171249 | ||
| Genetic reagent (Arabidopsis thaliana) | 35S::HSL3-GFP | This paper | Figure 3 and Materials and methods | |
| Genetic reagent (Arabidopsis thaliana) | pHSL3::BIR3-HSL3-FLAG | This paper | Chimera created using the method from https://doi.org/10.1105/tpc.20.00138 | |
| Cell line (Trichoplusia ni) | Tnao38 | https://doi.org/10.1186/1472-6750-12-12 | Cell line maintained in J Santiago lab | |
| Antibody | Anti-BAK1 (rabbit polyclonal) | https://doi.org/10.1105/tpc.111.084301 | WB (1:2000) | |
| Antibody | Anti-GFP (HRP-conjugated mouse monoclonal) | Santa Cruz | sc-9996 | WB (1:5000) |
| Antibody | Anti-pMAPK (rabbit polyclonal) | Cell Signaling | p44/42 MAPK (Erk1/2) antibody #9,102 | WB (1:4000) |
| Recombinant DNA reagent | 35S::HSL3-GFP (plasmid) | This paper | Figure 3 | |
| Recombinant DNA reagent | pHSL3::BIR3-HSL3-FLAG (plasmid) | This paper | Used to generate transgenic plants in Figure 3 | |
| Recombinant DNA reagent | pHSL3::LTI6B-Citrine (plasmid) | This paper | Used to generate transgenic plants in Figure 3—figure supplement 4 | |
| Recombinant DNA reagent | pHSL3::BIR3F146A,R170A -HSL3-Citrine (plasmid) | This paper | Used to generate transgenic plants in Figure 3—figure supplement 4 | |
| Recombinant DNA reagent | pHSL3::BIR3-HSL3-CITRINE (plasmid) | This paper | Used to generate transgenic plants in Figure 3—figure supplement 4 | |
| Commercial assay or kit | GFP-Trap | Chromotek | Cat. #: gta-20 | |
| Software, algorithm | GraphPad | GraphPad software |
Additional files
-
Supplementary file 1
Transcripts predicted to encode proteins <150 amino acids up-regulated following 1 μM flg22 treatment for 90 min (Bjornson et al., 2021).
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp1-v2.xlsx
-
Supplementary file 2
Spectral counts of peptides identified through affinity purification of the BAK1 complex.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp2-v2.xlsx
-
Supplementary file 3
Differential gene expression induced by 30 min CTNIP448-70 treatment.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp3-v2.csv
-
Supplementary file 4
Gene ontology enrichment following 30 min CTNIP448-70 treatment.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp4-v2.csv
-
Supplementary file 5
Primers used in this study.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp5-v2.xlsx
-
Supplementary file 6
Synthetic peptides used in this study.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp6-v2.docx
-
Supplementary file 7
Species included in CTNIP and RK search.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp7-v2.zip
-
Supplementary file 8
Initial CTNIP candidates used for the search.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp8-v2.fasta
-
Supplementary file 9
Identified CTNIPs relaxed.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp9-v2.zip
-
Supplementary file 10
Identified CTNIPs confident.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp10-v2.zip
-
Supplementary file 11
Initial RK candidates used for the search.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp11-v2.fasta
-
Supplementary file 12
Alignment of RKs identified.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp12-v2.zip
-
Supplementary file 13
Receptor phylogeny.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp13-v2.zip
-
Supplementary file 14
Full-length alignment of HAE/HSL/CEPR/RLK7/IKU2 clade.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp14-v2.zip
-
Supplementary file 15
Full-length HAE/HSL/CEPR/RLK7/IKU2 clade phylogeny.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp15-v2.zip
-
Supplementary file 16
Leucine-rich repeat (LRR) domain alignment of HAE/HSL/CEPR/RLK7/IKU2 clade.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp16-v2.zip
-
Supplementary file 17
Leucine-rich repeat (LRR) domain HAE/HSL/CEPR/RLK7/IKU2 clade phylogeny.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp17-v2.zip
-
Supplementary file 18
Kinase domain alignment of HAE/HSL/CEPR/RLK7/IKU2 clade.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp18-v2.zip
-
Supplementary file 19
Kinase domain HAE/HSL/CEPR/RLK7/IKU2 clade phylogeny.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp19-v2.zip
-
Supplementary file 20
Full-length HAESA-LIKE 3 (HSL3) alignment.
- https://cdn.elifesciences.org/articles/74687/elife-74687-supp20-v2.zip
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/74687/elife-74687-transrepform1-v2.docx
